Treatment of cancer using a cd33 chimeric antigen receptor

ABSTRACT

The invention provides compositions and methods for treating diseases associated with expression of CD33. The invention also relates to chimeric antigen receptor (CAR) specific to CD33, vectors encoding the same, and recombinant T cells comprising the CD33 CAR. The invention also includes methods of administering a genetically modified T cell expressing a CAR that comprises a CD33 binding domain.

This application is a divisional of U.S. application Ser. No.15/689,163, filed Aug. 29, 2017, which is a divisional of U.S.application Ser. No. 14/805,236, filed Jul. 21, 2015, now allowed, whichclaims priority to PCT Application No. PCT/CN2014/082589, filed Jul. 21,2014, and PCT Application No. PCT/CN2014/090504, filed Nov. 6, 2014. Theentire contents of these applications are incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 30, 2015, isnamed N2067-704710_SL.txt and is 322,538 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to the use of immune effectorcells (e.g., T cells, NK cells) engineered to express a Chimeric AntigenReceptor (CAR) to treat a disease associated with expression of theCluster of Differentiation 33 protein (CD33).

BACKGROUND OF THE INVENTION

Most patients with acute myeloid leukemia (AML) are incurable usingstandard therapy (Mrozek et al, 2012, J Clin Oncol, 30:4515-23) andthose with relapsed or refractory AML (RR-AML) have a particularly poorprognosis (Kern et al, 2003, Blood 2003, 101:64-70; Wheatley et al,1999, Br J Haematol, 107:69-79).

Genetic engineering can impart to T cells specificity toward a target ofchoice. T cells can be transduced with genetic material encoding asingle chain variable fragment (scFv) of an antibody, in conjunctionwith a signaling molecule, thereby using the complementarity determiningregion (CDR) to recognize a cell surface antigen in a non-MHC restrictedmanner. These cells are termed chimeric antigen receptor (CAR) T cells.Preclinical and clinical attempts to target at least 20 differentsurface molecules in a variety of malignancies have shown some activity,yet these attempts were often limited by poor persistence of the infusedCART cell product (Sadelain et al, 2009, Curr Opin Immunol 2009,21:215-23). Recent success with anti-CD19 redirected T cells in patientswith advanced chronic lymphoid leukemia (CLL) and acute lymphoidleukemia (ALL) (Porter et al, 2011, N Engl J Med, 365:725-33; Kalos etal, 2011, Science Transl Med, 3:95ra73; Grupp and Kalos, 2013, N Engl JMed, 368:1509-18) demonstrated that these cells can eradicate massivetumor burden after a single infusion with remission lasting up to 3years to date, underscoring the dramatic potential of CAR T celltherapy. There have been few preclinical attempts to target AML inanimal models (Marin et al, 2010, Haematologica, 95:2144-52; Tettamantiet al, 2013, Br J Haematol, 161:389-401). A recently published smallclinical trial demonstrated that it is feasible to produce and infuse Tcells to patients with an aggressive malignancy (Ritchie et al, 2013,Mol Ther, 2013 November; 21(11):2122-9). Besides the ability for thechimeric antigen receptor on the genetically modified T cells torecognize and destroy the targeted cells, a successful therapeutic Tcell therapy needs to have the ability to proliferate and persist overtime, and to further monitor for leukemic cell escapees. The variablequality of T cells whether it is a result of anergy, suppression orexhaustion can have effects on CAR-transformed T cells' performance.Skilled practitioners have limited control over the variability in thequality of T cells at this time. To be effective, CAR transformedpatient T cells need to persist and maintain the ability to proliferatein response to the CAR's antigen. It has been shown that T cells fromALL patient can do this with CART19 comprising a murine scFv (see, e.g.,Grupp et al., NEJM 368:1509-1518 (2013)).

SUMMARY OF THE INVENTION

In a first aspect, the invention features an isolated nucleic acidmolecule encoding a chimeric antigen receptor (CAR), wherein the CARcomprises an antibody or antibody fragment which includes a CD33 bindingdomain (e.g., a human or humanized CD33 binding domain), a transmembranedomain, and an intracellular signaling domain (e.g., an intracellularsignaling domain comprising a costimulatory domain and/or a primarysignaling domain). In one embodiment, the CAR comprises an antibody orantibody fragment which includes a CD33 binding domain described herein(e.g., a human or humanized CD33 binding domain described herein), atransmembrane domain described herein, and an intracellular signalingdomain described herein (e.g., an intracellular signaling domaincomprising a costimulatory domain and/or a primary signaling domain).

In one embodiment, the encoded CD33 binding domain comprises one or more(e.g., all three) light chain complementary determining region 1 (LCCDR1), light chain complementary determining region 2 (LC CDR2), andlight chain complementary determining region 3 (LC CDR3) of a CD33binding domain described herein, and/or one or more (e.g., all three)heavy chain complementary determining region 1 (HC CDR1), heavy chaincomplementary determining region 2 (HC CDR2), and heavy chaincomplementary determining region 3 (HC CDR3) of a CD33 binding domaindescribed herein, e.g., a CD33 binding domain comprising one or more,e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. Inone embodiment, the encoded CD33 binding domain (e.g., a human orhumanized CD33 binding domain) comprises a light chain variable regiondescribed herein (e.g., in Table 2 or 9) and/or a heavy chain variableregion described herein (e.g., in Table 2 or 9). In one embodiment, theencoded CD33 binding domain is a scFv comprising a light chain and aheavy chain of an amino acid sequence of Table 2 or 9. In an embodiment,the encoded CD33 binding domain (e.g., an scFv) comprises a light chainvariable region comprising an amino acid sequence having at least one,two or three modifications (e.g., substitutions, e.g., conservativesubstitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions e.g., conservative substitutions) of an amino acidsequence of a light chain variable region provided in Table 2 or 9, or asequence with 95-99% identity with an amino acid sequence of Table 2 or9; and/or a heavy chain variable region comprising an amino acidsequence having at least one, two or three modifications (e.g.,substitutions, e.g., conservative substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions, e.g., conservativesubstitutions) of an amino acid sequence of a heavy chain variableregion provided in Table 2 or 9, or a sequence with 95-99% identity toan amino acid sequence of Table 2 or 9.

In other embodiments, the encoded CD33 binding domain comprises a HCCDR1, a HC CDR2, and a HC CDR3 of any CD33 heavy chain binding domainamino acid sequences listed in Table 2 or 9. In embodiments, the CD33binding domain further comprises a LC CDR1, a LC CDR2, and a LC CDR3. Inembodiments, the CD33 binding domain comprises a LC CDR1, a LC CDR2, anda LC CDR3 of any CD33 light chain binding domain amino acid sequenceslisted in Table 2 or 9.

In some embodiments, the encoded CD33 binding domain comprises one, twoor all of LC CDR1, LC CDR2, and LC CDR3 of any CD33 light chain bindingdomain amino acid sequences listed in Table 2 or 9, and one, two or allof HC CDR1, HC CDR2, and HC CDR3 of any CD33 heavy chain binding domainamino acid sequences listed in Table 2 or 9.

In one embodiment, the encoded CD33 binding domain comprises an aminoacid sequence sequence selected from a group consisting of SEQ IDNO:39-47, 57-65, 66-74, or 262-268. In an embodiment, the encoded CD33binding domain (e.g., an scFv) comprises an amino acid sequence havingat least one, two or three modifications (e.g., substitutions, e.g.,conservative substitutions) but not more than 30, 20 or 10 modifications(e.g., substitutions, e.g., conservative substitutions) of an amino acidsequence of SEQ ID NO:39-47, 57-65, 66-74, or 262-268, or a sequencewith 95-99% identity with an amino acid sequence of SEQ ID NO:39-47,57-65, 66-74, or 262-268. In another embodiment, the encoded CD33binding domain comprises a heavy chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NO:57-65, or a sequence with 95-99% identity thereof. In anotherembodiment, the encoded CD33 binding domain comprises a light chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 66-74, or a sequence with 95-99% identitythereof. In one embodiment, the nucleic acid molecule comprises anucleotide sequence selected from the group consisting of SEQ ID NO:255-261, or a sequence with 95-99% identity thereof.

In one embodiment, the encoded CD33 binding domain includes a(Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4(SEQ ID NO:26). The light chain variable region and heavy chain variableregion of a scFv can be, e.g., in any of the following orientations:light chain variable region-linker-heavy chain variable region or heavychain variable region-linker-light chain variable region.

In one embodiment, the encoded CAR includes a transmembrane domain thatcomprises a transmembrane domain of a protein, e.g., a protein describedherein, e.g., selected from the group consisting of the alpha, beta orzeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 andCD154. In one embodiment, the encoded transmembrane domain comprises thesequence of SEQ ID NO: 6. In one embodiment, the encoded transmembranedomain comprises an amino acid sequence comprising at least one, two orthree modifications, but not more than 20, 10 or 5 modifications of theamino acid sequence of SEQ ID NO:6, or a sequence with 95-99% identityto an amino acid sequence of SEQ ID NO:6. In one embodiment, the nucleicacid sequence encoding the transmembrane domain comprises the sequenceof SEQ ID NO: 17, or a sequence with 95-99% identity thereof.

In one embodiment, the encoded CD33 binding domain is connected to thetransmembrane domain by a hinge region, e.g., a hinge region describedherein. In one embodiment, the encoded hinge region comprises SEQ IDNO:2, or a sequence with 95-99% identity thereof. In one embodiment, thenucleic acid sequence encoding the hinge region comprises the sequenceof SEQ ID NO: 13, or a sequence with 95-99% identity thereof.

In one embodiment, the isolated nucleic acid molecule further comprisesa sequence encoding a costimulatory domain, e.g., a costimulatory domaindescribed herein. In embodiments, the intracellular signaling domaincomprises a costimulatory domain. In embodiments, the intracellularsignaling domain comprises a primary signaling domain. In embodiments,the intracellular signaling domain comprises a costimulatory domain anda primary signaling domain.

In one embodiment, the encoded costimulatory domain is a functionalsignaling domain obtained from a protein, e.g., described herein, e.g.,selected from the group consisting of MHC class I molecule, TNF receptorproteins, Immunoglobulin-like proteins, cytokine receptors, integrins,signaling lymphocytic activation molecules (SLAM proteins), activatingNK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27,CD28, CD30, CD40, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3,CD5, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.In embodiments, the encoded costimulatory domain comprises 4-1BB, CD27,CD28, or ICOS.

In one embodiment, the encoded costimulatory domain of 4-1BB comprisesthe amino acid sequence of SEQ ID NO:7. In one embodiment, the encodedcostimulatory domain comprises an amino acid sequence having at leastone, two or three modifications but not more than 20, 10 or 5modifications of an amino acid sequence of SEQ ID NO:7, or a sequencewith 95-99% identity to an amino acid sequence of SEQ ID NO:7. In oneembodiment, the nucleic acid sequence encoding the costimulatory domaincomprises the nucleotide sequence of SEQ ID NO:18, or a sequence with95-99% identity thereof. In another embodiment, the encodedcostimulatory domain of CD28 comprises the amino acid sequence of SEQ IDNO:379. In one embodiment, the encoded costimulatory domain comprises anamino acid sequence having at least one, two or three modifications butnot more than 20, 10 or 5 modifications of an amino acid sequence of SEQID NO:379, or a sequence with 95-99% identity to an amino acid sequenceof SEQ ID NO:379. In one embodiment, the nucleic acid sequence encodingthe costimulatory domain of CD28 comprises the nucleotide sequence ofSEQ ID NO:380, or a sequence with 95-99% identity thereof. In anotherembodiment, the encoded costimulatory domain of CD27 comprises the aminoacid sequence of SEQ ID NO:8. In one embodiment, the encodedcostimulatory domain comprises an amino acid sequence having at leastone, two or three modifications but not more than 20, 10 or 5modifications of an amino acid sequence of SEQ ID NO:8, or a sequencewith 95-99% identity to an amino acid sequence of SEQ ID NO:8. In oneembodiment, the nucleic acid sequence encoding the costimulatory domainof CD27 comprises the nucleotide sequence of SEQ ID NO:19, or a sequencewith 95-99% identity thereof. In another embodiment, the encodedcostimulatory domain of ICOS comprises the amino acid sequence of SEQ IDNO:381. In one embodiment, the encoded costimulatory domain of ICOScomprises an amino acid sequence having at least one, two or threemodifications but not more than 20, 10 or 5 modifications of an aminoacid sequence of SEQ ID NO:381, or a sequence with 95-99% identity to anamino acid sequence of SEQ ID NO:381. In one embodiment, the nucleicacid sequence encoding the costimulatory domain of ICOS comprises thenucleotide sequence of SEQ ID NO:382, or a sequence with 95-99% identitythereof.

In embodiments, the encoded primary signaling domain comprises afunctional signaling domain of CD3 zeta. In embodiments, the functionalsignaling domain of CD3 zeta comprises the amino acid sequence of SEQ IDNO: 9 (mutant CD3 zeta) or SEQ ID NO: 10 (wild type human CD3 zeta), ora sequence with 95-99% identity thereof.

In one embodiment, the encoded intracellular signaling domain comprisesa functional signaling domain of 4-1BB and/or a functional signalingdomain of CD3 zeta. In one embodiment, the encoded intracellularsignaling domain of 4-1BB comprises the amino acid sequence of SEQ IDNO: 7 and/or the CD3 zeta amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10. In one embodiment, the intracellular signaling domain comprisesan amino acid sequence having at least one, two or three modificationsbut not more than 20, 10 or 5 modifications of an amino acid sequence ofSEQ ID NO:7 and/or an amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10, or a sequence with 95-99% identity to an amino acid sequence ofSEQ ID NO:7 and/or an amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10. In one embodiment, the encoded intracellular signaling domaincomprises the sequence of SEQ ID NO:7 and the sequence of SEQ ID NO:9 orSEQ ID NO:10, wherein the sequences comprising the intracellularsignaling domain are expressed in the same frame and as a singlepolypeptide chain. In one embodiment, the nucleic acid sequence encodingthe intracellular signaling domain of 4-1BB comprises the nucleotidesequence of SEQ ID NO:18, or a sequence with 95-99% identity thereof,and/or the CD3 zeta nucleotide sequence of SEQ ID NO:20 or SEQ ID NO:21,or a sequence with 95-99% identity thereof.

In one embodiment, the encoded intracellular signaling domain comprisesa functional signaling domain of CD27 and/or a functional signalingdomain of CD3 zeta. In one embodiment, the encoded intracellularsignaling domain of CD27 comprises the amino acid sequence of SEQ ID NO:8 and/or the CD3 zeta amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10. In one embodiment, the intracellular signaling domain comprisesan amino acid sequence having at least one, two or three modificationsbut not more than 20, 10 or 5 modifications of an amino acid sequence ofSEQ ID NO:8 and/or an amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10, or a sequence with 95-99% identity to an amino acid sequence ofSEQ ID NO:8 and/or an amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10. In one embodiment, the encoded intracellular signaling domaincomprises the sequence of SEQ ID NO:8 and the sequence of SEQ ID NO:9 orSEQ ID NO:10, wherein the sequences comprising the intracellularsignaling domain are expressed in the same frame and as a singlepolypeptide chain. In one embodiment, the nucleic acid sequence encodingthe intracellular signaling domain of CD27 comprises the nucleotidesequence of SEQ ID NO:19, or a sequence with 95-99% identity thereof,and/or the CD3 zeta nucleotide sequence of SEQ ID NO:20 or SEQ ID NO:21,or a sequence with 95-99% identity thereof.

In one embodiment, the encoded intracellular signaling domain comprisesa functional signaling domain of CD28 and/or a functional signalingdomain of CD3 zeta. In one embodiment, the encoded intracellularsignaling domain of CD28 comprises the amino acid sequence of SEQ ID NO:379 and/or the CD3 zeta amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10. In one embodiment, the intracellular signaling domain comprisesan amino acid sequence having at least one, two or three modificationsbut not more than 20, 10 or 5 modifications of an amino acid sequence ofSEQ ID NO:379 and/or an amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10, or a sequence with 95-99% identity to an amino acid sequence ofSEQ ID NO:379 and/or an amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10. In one embodiment, the encoded intracellular signaling domaincomprises the sequence of SEQ ID NO:379 and the sequence of SEQ ID NO:9or SEQ ID NO:10, wherein the sequences comprising the intracellularsignaling domain are expressed in the same frame and as a singlepolypeptide chain. In one embodiment, the nucleic acid sequence encodingthe intracellular signaling domain of CD28 comprises the nucleotidesequence of SEQ ID NO:380, or a sequence with 95-99% identity thereof,and/or the CD3 zeta nucleotide sequence of SEQ ID NO:20 or SEQ ID NO:21,or a sequence with 95-99% identity thereof.

In one embodiment, the encoded intracellular signaling domain comprisesa functional signaling domain of ICOS and/or a functional signalingdomain of CD3 zeta. In one embodiment, the encoded intracellularsignaling domain of ICOS comprises the amino acid sequence of SEQ ID NO:381 and/or the CD3 zeta amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10. In one embodiment, the intracellular signaling domain comprisesan amino acid sequence having at least one, two or three modificationsbut not more than 20, 10 or 5 modifications of an amino acid sequence ofSEQ ID NO:381 and/or an amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10, or a sequence with 95-99% identity to an amino acid sequence ofSEQ ID NO:381 and/or an amino acid sequence of SEQ ID NO:9 or SEQ IDNO:10. In one embodiment, the encoded intracellular signaling domaincomprises the sequence of SEQ ID NO:381 and the sequence of SEQ ID NO:9or SEQ ID NO:10, wherein the sequences comprising the intracellularsignaling domain are expressed in the same frame and as a singlepolypeptide chain. In one embodiment, the nucleic acid sequence encodingthe intracellular signaling domain of ICOS comprises the nucleotidesequence of SEQ ID NO:382, or a sequence with 95-99% identity thereof,and/or the CD3 zeta nucleotide sequence of SEQ ID NO:20 or SEQ ID NO:21,or a sequence with 95-99% identity thereof.

In another aspect, the invention pertains to an isolated nucleic acidmolecule encoding a CAR construct comprising a leader sequence, e.g., aleader sequence described herein, e.g., the amino acid sequence of SEQID NO: 1; a CD33 binding domain described herein, e.g., a CD33 bindingdomain comprising a LC CDR1, a LC CDR2, a LC CDR3, a HC CDR1, a HC CDR2and a HC CDR3 described herein (e.g., a human or humanized CD33 bindingdomain described in Table 2 or 9), or a sequence with 95-99% identifythereof; a hinge region described herein, e.g., the amino acid sequenceof SEQ ID NO:2; a transmembrane domain described herein, e.g., having asequence of SEQ ID NO: 6; and an intracellular signaling domain, e.g.,an intracellular signaling domain described herein. In one embodiment,the encoded intracellular signaling domain comprises a costimulatorydomain, e.g., a costimulatory domain described herein (e.g., a 4-1BBcostimulatory domain having the amino acid sequence of SEQ ID NO:7 or aCD27 costimulatory domain having the amino acid sequence of SEQ IDNO:8), and/or a primary signaling domain, e.g., a primary signalingdomain described herein, (e.g., a CD3 zeta stimulatory domain having asequence of SEQ ID NO:9 or SEQ ID NO:10). In one embodiment, theisolated nucleic acid molecule encoding the CAR construct includes aleader sequence encoded by the nucleic acid sequence of SEQ ID NO:1, ora sequence with 95-99% identity thereto.

In one embodiment, the isolated nucleic acid molecule comprises (e.g.,consists of) a nucleic acid encoding a CAR amino acid sequence of SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56; or an aminoacid having one, two or three modifications (e.g., substitutions) butnot more than 30, 20 or 10 modifications (e.g., substitutions, e.g.,conservative substitutions) of an amino acid sequence of SEQ ID NO: 48,SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56; or or an amino acidsequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to an aminoacid sequence of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQID NO: 56.

In one embodiment, the isolated nucleic acid molecule comprises (e.g.,consists of) a nucleic acid sequence of SEQ ID NO:75, SEQ ID NO:76, SEQID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ IDNO:82, or SEQ ID NO:83 or a nucleic acid sequence having 85%, 90%, 95%,96%, 97%, 98% or 99% identity to a nucleic acid sequence of SEQ IDNO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ IDNO:80, SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83.

In one aspect, the invention pertains to an isolated nucleic acidmolecule encoding a CD33 binding domain, wherein the CD33 binding domaincomprises one or more (e.g., all three) light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and/or light chain complementary determining region3 (LC CDR3) of a CD33 binding domain described herein, and one or more(e.g., all three) heavy chain complementary determining region 1 (HCCDR1), heavy chain complementary determining region 2 (HC CDR2), andheavy chain complementary determining region 3 (HC CDR3) of a CD33binding domain described herein, e.g., a human or humanized CD33 bindingdomain comprising one or more, e.g., all three, LC CDRs and one or more,e.g., all three, HC CDRs.

In other embodiments, the encoded CD33 binding domain comprises a HCCDR1, a HC CDR2, and a HC CDR3 of any CD33 heavy chain binding domainamino acid sequences listed in Table 2 or 9. In embodiments, the CD33binding domain further comprises a LC CDR1, a LC CDR2, and a LC CDR3. Inembodiments, the CD33 binding domain comprises a LC CDR1, a LC CDR2, anda LC CDR3 of any CD33 light chain binding domain amino acid sequenceslisted in Table 2 or 9.

In some embodiments, the encoded CD33 binding domain comprises one, twoor all of LC CDR1, LC CDR2, and LC CDR3 of any CD33 light chain bindingdomain amino acid sequences listed in Table 2 or 9, and one, two or allof HC CDR1, HC CDR2, and HC CDR3 of any CD33 heavy chain binding domainamino acid sequences listed in Table 2 or 9.

In one embodiment, the encoded CD33 binding domain comprises a lightchain variable region described herein (e.g., in SEQ ID NO:66, 67, 68,69, 70, 71, 72, 73, or 74) and/or a heavy chain variable regiondescribed herein (e.g., in SEQ ID NO:57, 58, 59, 60, 61, 62, 63, 64, or65). In one embodiment, the encoded CD33 binding domain is a scFvcomprising a light chain and a heavy chain of an amino acid sequence ofin SEQ ID NO:39, 40, 41, 42, 43, 44, 45, 46, or 47. In an embodiment,the CD33 binding domain (e.g., an scFv) comprises: a light chainvariable region comprising an amino acid sequence having at least one,two or three modifications (e.g., substitutions, e.g., conservativesubstitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions, e.g., conservative substitutions) of an amino acidsequence of a light chain variable region provided in SEQ ID NO: 66, 67,68, 69, 70, 71, 72, 73, or 74, or a sequence with 95-99% identity withan amino acid sequence of SEQ ID NO: 66, 67, 68, 69, 70, 71, 72, 73, or74; and/or a heavy chain variable region comprising an amino acidsequence having at least one, two or three modifications (e.g.,substitutions, e.g., conservative substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions, e.g., conservativesubstitutions) of an amino acid sequence of a heavy chain variableregion provided in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64, or 65, ora sequence with 95-99% identity to an amino acid sequence in SEQ ID NO:57, 58, 59, 60, 61, 62, 63, 64, or 65. In one embodiment, the CD33binding domain comprises a sequence selected from a group consisting ofSEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43,SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, or asequence with 95-99% identify thereof. In one embodiment, the encodedCD33 binding domain is a scFv, and a light chain variable regioncomprising an amino acid sequence described herein, e.g., in Table 2, isattached to a heavy chain variable region comprising an amino acidsequence described herein, e.g., in Table 2, via a linker, e.g., alinker described herein. In one embodiment, the encoded CD33 bindingdomain includes a (Gly₄-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6,preferably 4 (SEQ ID NO: 26). The light chain variable region and heavychain variable region of a scFv can be, e.g., in any of the followingorientations: light chain variable region-linker-heavy chain variableregion or heavy chain variable region-linker-light chain variableregion.

In another aspect, the invention pertains to an isolated CD33 bindingdomain (e.g., a polypeptide, antibody or fragment thereof) moleculeencoded by the nucleic acid molecule. In one embodiment, the isolatedCD33 binding domain comprises a sequence selected from the groupconsisting of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:56,or a sequence with 95-99% identify thereof.

In another aspect, the invention pertains to an isolated chimericantigen receptor (CAR) molecule (e.g., polypeptide) comprising a CD33binding domain (e.g., a human or humanized antibody or antibody fragmentthat specifically binds to CD33), a transmembrane domain, and anintracellular signaling domain (e.g., an intracellular signaling domaincomprising a costimulatory domain and/or a primary signaling domain). Inone embodiment, the CAR comprises an antibody or antibody fragment whichincludes a CD33 binding domain described herein (e.g., a human orhumanized antibody or antibody fragment that specifically binds to CD33as described herein), a transmembrane domain described herein, and anintracellular signaling domain described herein (e.g., an intracellularsignaling domain comprising a costimulatory domain and/or a primarysignaling domain described herein).

In one embodiment, the CD33 binding domain comprises one or more (e.g.,all three) light chain complementary determining region 1 (LC CDR1),light chain complementary determining region 2 (LC CDR2), and lightchain complementary determining region 3 (LC CDR3) of a CD33 bindingdomain described herein, and one or more (e.g., all three) heavy chaincomplementary determining region 1 (HC CDR1), heavy chain complementarydetermining region 2 (HC CDR2), and/or heavy chain complementarydetermining region 3 (HC CDR3) of a CD33 binding domain describedherein, e.g., a CD33 binding domain comprising one or more, e.g., allthree, LC CDRs and one or more, e.g., all three, HC CDRs. In oneembodiment, the CD33 binding domain comprises a light chain variableregion described herein (e.g., in Table 2) and/or a heavy chain variableregion described herein (e.g., in Table 2 or 9). In one embodiment, theCD33 binding domain is a scFv comprising a light chain and a heavy chainof an amino acid sequence listed in Table 2 or 9. In an embodiment, theCD33 binding domain (e.g., an scFv) comprises: a light chain variableregion comprising an amino acid sequence having at least one, two orthree modifications (e.g., substitutions, e.g., conservativesubstitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions, e.g., conservative substitutions) of an amino acidsequence of a light chain variable region provided in Table 2 or 9, or asequence with 95-99% identity with an amino acid sequence provided inTable 2 or 9; and/or a heavy chain variable region comprising an aminoacid sequence having at least one, two or three modifications (e.g.,substitutions, e.g., conservative substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions, e.g., conservativesubstitutions) of an amino acid sequence of a heavy chain variableregion provided in Table 2 or 9, or a sequence with 95-99% identity toan amino acid sequence provided in Table 2 or 9.

In other embodiments, the CD33 binding domain comprises a HC CDR1, a HCCDR2, and a HC CDR3 of any CD33 heavy chain binding domain amino acidsequences listed in Table 2 or 9. In embodiments, the CD33 bindingdomain further comprises a LC CDR1, a LC CDR2, and a LC CDR3. Inembodiments, the CD33 binding domain comprises a LC CDR1, a LC CDR2, anda LC CDR3)\ of any CD33 light chain binding domain amino acid sequenceslisted in Table 2 or 9.

In some embodiments, the CD33 binding domain comprises one, two or allof LC CDR1, LC CDR2, and LC CDR3 of any CD33 light chain binding domainamino acid sequences listed in Table 2 or 9, and one, two or all of HCCDR1, HC CDR2, and HC CDR3 of any CD33 heavy chain binding domain aminoacid sequences listed in Table 2 or 9.

In one embodiment, the CD33 binding domain comprises a sequence selectedfrom a group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:57-74, or SEQ ID NO:262-268; or am amino acid sequencehaving at least one, two or three modifications (e.g., substitutions,e.g., conservative substitutions) but not more than 30, 20 or 10modifications (e.g., substitutions, e.g., conservative substitutions) toany of the aforesaid sequences; or a sequence with 95-99% identify toany of the aforesaid sequences. In one embodiment, the CD33 bindingdomain is a scFv, and a light chain variable region comprising an aminoacid sequence described herein, e.g., in Table 2 or 9, is attached to aheavy chain variable region comprising an amino acid sequence describedherein, e.g., in Table 2 or 9, via a linker, e.g., a linker describedherein. In one embodiment, the CD33 binding domain includes a(Gly₄-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 4 (SEQID NO: 26). The light chain variable region and heavy chain variableregion of a scFv can be, e.g., in any of the following orientations:light chain variable region-linker-heavy chain variable region or heavychain variable region-linker-light chain variable region.

In one embodiment, the isolated CAR molecule comprises a transmembranedomain of a protein, e.g., a protein described herein, e.g., selectedfrom the group consisting of the the alpha, beta or zeta chain of theT-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16,CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In oneembodiment, the transmembrane domain comprises a sequence of SEQ ID NO:6. In one embodiment, the transmembrane domain comprises an amino acidsequence having at least one, two or three modifications (e.g.,substitutions, e.g., conservative substitutions) but not more than 20,10 or 5 modifications (e.g., substitutions, e.g., conservativesubstitutions) of an amino acid sequence of SEQ ID NO: 6, or a sequencewith 95-99% identity to an amino acid sequence of SEQ ID NO: 6.

In one embodiment, the CD33 binding domain is connected to thetransmembrane domain by a hinge region, e.g., a hinge region describedherein. In one embodiment, the encoded hinge region comprises SEQ IDNO:2, or a sequence with 95-99% identity thereof.

In one embodiment, the isolated CAR molecule further comprises asequence encoding a costimulatory domain, e.g., a costimulatory domaindescribed herein.

In embodiments, the intracellular signaling domain of the isolated CARmolecule comprises a costimulatory domain. In embodiments, theintracellular signaling domain of the isolated CAR molecule comprises aprimary signaling domain. In embodiments, the intracellular signalingdomain of the isolated CAR molecule comprises a costimulatory domain anda primary signaling domain.

In one embodiment, the costimulatory domain comprises a functionalsignaling domain of a protein selected from the group consisting of MHCclass I molecule, TNF receptor proteins, Immunoglobulin-like proteins,cytokine receptors, integrins, signaling lymphocytic activationmolecules (SLAM proteins), activating NK cell receptors, BTLA, a Tollligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD5, ICAM-1,LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CD5, ICAM-1, ICOS (CD278),GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44,NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7Ralpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f,ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX,CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2,TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69,SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligandthat specifically binds with CD83.

In one embodiment, the costimulatory domain of 4-1BB comprises the aminoacid sequence of SEQ ID NO:7. In one embodiment, the costimulatorydomain comprises an amino acid sequence having at least one, two orthree modifications (e.g., substitutions, e.g., conservativesubstitutions) but not more than 20, 10 or 5 modifications (e.g.,substitutions, e.g., conservative substitutions) of an amino acidsequence of SEQ ID NO:7, or a sequence with 95-99% identity to an aminoacid sequence of SEQ ID NO:7. In another embodiment, the costimulatorydomain of CD28 comprises the amino acid sequence of SEQ ID NO:379. Inone embodiment, the costimulatory domain comprises an amino acidsequence having at least one, two or three modifications but not morethan 20, 10 or 5 modifications of an amino acid sequence of SEQ IDNO:379, or a sequence with 95-99% identity to an amino acid sequence ofSEQ ID NO:379. In another embodiment, the costimulatory domain of CD27comprises the amino acid sequence of SEQ ID NO:8. In one embodiment, thecostimulatory domain comprises an amino acid sequence having at leastone, two or three modifications but not more than 20, 10 or 5modifications of an amino acid sequence of SEQ ID NO:8, or a sequencewith 95-99% identity to an amino acid sequence of SEQ ID NO:8. Inanother embodiment, the costimulatory domain of ICOS comprises the aminoacid sequence of SEQ ID NO:381. In one embodiment, the costimulatorydomain comprises an amino acid sequence having at least one, two orthree modifications but not more than 20, 10 or 5 modifications of anamino acid sequence of SEQ ID NO:381, or a sequence with 95-99% identityto an amino acid sequence of SEQ ID NO:381.

In embodiments, the primary signaling domain comprises a functionalsignaling domain of CD3 zeta. In embodiments, the functional signalingdomain of CD3 zeta comprises SEQ ID NO: 9 (mutant CD3 zeta) or SEQ IDNO: 10 (wild type human CD3 zeta), or a sequence with 95-99% identitythereof.

In one embodiment, the intracellular signaling domain comprises afunctional signaling domain of 4-1BB and/or a functional signalingdomain of CD3 zeta. In one embodiment, the intracellular signalingdomain comprises the amino acid sequence of SEQ ID NO: 7 and/or theamino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In one embodiment,the intracellular signaling domain comprises an amino acid sequencehaving at least one, two or three modifications (e.g., substitutions,e.g., conservative substitutions) but not more than 20, 10 or 5modifications (e.g., substitutions, e.g., conservative substitutions) ofan amino acid sequence of SEQ ID NO: 7 and/or the sequence of SEQ IDNO:9 or SEQ ID NO:10, or a sequence with 95-99% identity to an aminoacid sequence of SEQ ID NO: 7 and/or the sequence of SEQ ID NO:9 or SEQID NO:10. In one embodiment, the intracellular signaling domaincomprises the amino acid sequence of SEQ ID NO: 7 and/or the sequence ofSEQ ID NO:9 or SEQ ID NO:10, wherein the sequences comprising theintracellular signaling domain are expressed in the same frame and as asingle polypeptide chain.

In one embodiment, the intracellular signaling domain comprises afunctional signaling domain of CD27 and/or a functional signaling domainof CD3 zeta. In one embodiment, the intracellular signaling domain ofCD27 comprises the amino acid sequence of SEQ ID NO: 8 and/or the CD3zeta amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In oneembodiment, the intracellular signaling domain comprises an amino acidsequence having at least one, two or three modifications but not morethan 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:8and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, or asequence with 95-99% identity to an amino acid sequence of SEQ ID NO:8and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In oneembodiment, the intracellular signaling domain comprises the sequence ofSEQ ID NO:8 and the sequence of SEQ ID NO:9 or SEQ ID NO:10, wherein thesequences comprising the intracellular signaling domain are expressed inthe same frame and as a single polypeptide chain.

In one embodiment, the intracellular signaling domain comprises afunctional signaling domain of CD28 and/or a functional signaling domainof CD3 zeta. In one embodiment, the encoded intracellular signalingdomain of CD28 comprises the amino acid sequence of SEQ ID NO: 379and/or the CD3 zeta amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.In one embodiment, the intracellular signaling domain comprises an aminoacid sequence having at least one, two or three modifications but notmore than 20, 10 or 5 modifications of an amino acid sequence of SEQ IDNO: 379 and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, ora sequence with 95-99% identity to an amino acid sequence of SEQ ID NO:379 and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In oneembodiment, the intracellular signaling domain comprises the sequence ofSEQ ID NO: 379 and the sequence of SEQ ID NO:9 or SEQ ID NO:10, whereinthe sequences comprising the intracellular signaling domain areexpressed in the same frame and as a single polypeptide chain.

In one embodiment, the intracellular signaling domain comprises afunctional signaling domain of ICOS and/or a functional signaling domainof CD3 zeta. In one embodiment, the intracellular signaling domain ofICOS comprises the amino acid sequence of SEQ ID NO: 381 and/or the CD3zeta amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In oneembodiment, the intracellular signaling domain comprises an amino acidsequence having at least one, two or three modifications but not morethan 20, 10 or 5 modifications of an amino acid sequence of SEQ IDNO:381 and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, ora sequence with 95-99% identity to an amino acid sequence of SEQ IDNO:381 and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. Inone embodiment, the encoded intracellular signaling domain comprises thesequence of SEQ ID NO:381 and the sequence of SEQ ID NO:9 or SEQ IDNO:10, wherein the sequences comprising the intracellular signalingdomain are expressed in the same frame and as a single polypeptidechain.

In one embodiment, the isolated CAR molecule further comprises a leadersequence, e.g., a leader sequence described herein. In one embodiment,the leader sequence comprises an amino acid sequence of SEQ ID NO: 1, ora sequence with 95-99% identity to an amino acid sequence of SEQ IDNO:1.

In another aspect, the invention pertains to an isolated CAR moleculecomprising a leader sequence, e.g., a leader sequence described herein,e.g., a leader sequence of SEQ ID NO: 1, or having 95-99% identitythereof, a CD33 binding domain described herein, e.g., a CD33 bindingdomain comprising a LC CDR1, a LC CDR2, a LC CDR3, a HC CDR1, a HC CDR2and a HC CDR3 described herein, e.g., a CD33 binding domain described inTable 2, or a sequence with 95-99% identify thereof, a hinge region,e.g., a hinge region described herein, e.g., a hinge region of SEQ IDNO:2, or having 95-99% identity thereof, a transmembrane domain, e.g., atransmembrane domain described herein, e.g., a transmembrane domainhaving a sequence of SEQ ID NO: 6 or a sequence having 95-99% identitythereof, an intracellular signaling domain, e.g., an intracellularsignaling domain described herein (e.g., an intracellular signalingdomain comprising a costimulatory domain and/or a primary signalingdomain). In one embodiment, the intracellular signaling domain comprisesa costimulatory domain, e.g., a costimulatory domain described herein,e.g., a 4-1BB costimulatory domain having a sequence of SEQ ID NO:7, orhaving 95-99% identity thereof, and/or a primary signaling domain, e.g.,a primary signaling domain described herein, e.g., a CD3 zetastimulatory domain having a sequence of SEQ ID NO:9 or SEQ ID NO:10, orhaving 95-99% identity thereof. In one embodiment, the intracellularsignaling domain comprises a costimulatory domain, e.g., a costimulatorydomain described herein, e.g., a 4-1BB costimulatory domain having asequence of SEQ ID NO:7, and/or a primary signaling domain, e.g., aprimary signaling domain described herein, e.g., a CD3 zeta stimulatorydomain having a sequence of SEQ ID NO:9 or SEQ ID NO:10.

In one embodiment, the isolated CAR molecule comprises (e.g., consistsof) an amino acid sequence of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, orSEQ ID NO:56, or an amino acid sequence having at least one, two, three,four, five, 10, 15, 20 or 30 modifications (e.g., substitutions, e.g.,conservative substitutions) but not more than 60, 50 or 40 modifications(e.g., substitutions, e.g., conservative substitutions) of an amino acidsequence of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, or SEQ ID NO:56, oran amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99%identity to an amino acid sequence of SEQ ID NO:48, SEQ ID NO:49, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:55, or SEQ ID NO:56.

In one aspect, the invention pertains to a CD33 binding domaincomprising one or more (e.g., all three) light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) of a CD33 binding domain described herein, and/or one or more(e.g., all three) heavy chain complementary determining region 1 (HCCDR1), heavy chain complementary determining region 2 (HC CDR2), andheavy chain complementary determining region 3 (HC CDR3) of a CD33binding domain described herein, e.g., a CD33 binding domain comprisingone or more, e.g., all three, LC CDRs and one or more, e.g., all three,HC CDRs.

In other embodiments, the CD33 binding domain comprises a HC CDR1, a HCCDR2, and a HC CDR3 of any CD33 heavy chain binding domain amino acidsequences listed in Table 2 or 9. In embodiments, the CD33 bindingdomain further comprises a LC CDR1, a LC CDR2, and a LC CDR3. Inembodiments, the CD33 binding domain comprises a LC CDR1, a LC CDR2, anda LC CDR3)\ of any CD33 light chain binding domain amino acid sequenceslisted in Table 2 or 9.

In some embodiments, the CD33 binding domain comprises one, two or allof LC CDR1, LC CDR2, and LC CDR3 of any CD33 light chain binding domainamino acid sequences listed in Table 2 or 9, and one, two or all of HCCDR1, HC CDR2, and HC CDR3 of any CD33 heavy chain binding domain aminoacid sequences listed in Table 2 or 9.

In one embodiment, the CD33 binding domain comprises a light chainvariable region described herein (e.g., in SEQ ID NO:66, 67, 68, 69, 70,71, 72, 73, or 74) and/or a heavy chain variable region described herein(e.g. in SEQ ID NO:57, 58, 59, 60, 61, 62, 63, 64, or 65). In oneembodiment, the CD33 binding domain is a scFv comprising a light chainand a heavy chain of an amino acid sequence of SEQ ID NO:39, 40, 41, 42,43, 44, 45, 46, or 47. In an embodiment, the CD33 binding domain (e.g.,an scFv) comprises: a light chain variable region comprising an aminoacid sequence having at least one, two or three modifications (e.g.,substitutions, e.g., conservative substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions, e.g., conservativesubstitutions) of an amino acid sequence of a light chain variableregion provided, in SEQ ID NO: 66, 67, 68, 69, 70, 71, 72, 73, or 74 ora sequence with 95-99% identity with an amino acid sequence in SEQ IDNO: 66, 67, 68, 69, 70, 71, 72, 73, or 74; and/or a heavy chain variableregion comprising an amino acid sequence having at least one, two orthree modifications (e.g., substitutions, e.g., conservativesubstitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions, e.g., conservative substitutions) of an amino acidsequence of a heavy chain variable region provided in SEQ ID NO: 57, 58,59, 60, 61, 62, 63, 64, or 65, or a sequence with 95-99% identity to anamino acid sequence in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64, or 65.In one embodiment, the CD33 binding domain comprises a sequence selectedfrom a group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, andSEQ ID NO:47, or a sequence with 95-99% identify thereof. In oneembodiment, the CD33 binding domain is a scFv, and a light chainvariable region comprising an amino acid sequence described herein,e.g., in Table 2 or 9, is attached to a heavy chain variable regioncomprising an amino acid sequence described herein, e.g., in Table 2 or9, via a linker, e.g., a linker described herein. In one embodiment, theCD33 binding domain includes a (Gly₄-Ser)n linker, wherein n is 1, 2, 3,4, 5, or 6, preferably 4 (SEQ ID NO: 26). The light chain variableregion and heavy chain variable region of a scFv can be, e.g., in any ofthe following orientations: light chain variable region-linker-heavychain variable region or heavy chain variable region-linker-light chainvariable region.

In another aspect, the invention pertains to a vector comprising anucleic acid molecule described herein, e.g., a nucleic acid moleculeencoding a CAR described herein. In one embodiment, the vector isselected from the group consisting of a DNA, a RNA, a plasmid, alentivirus vector, adenoviral vector, or a retrovirus vector.

In one embodiment, the vector is a lentivirus vector. In one embodiment,the vector further comprises a promoter. In one embodiment, the promoteris an EF-1 promoter. In one embodiment, the EF-1 promoter comprises asequence of SEQ ID NO: 11. In another embodiment, the promoter is a PGKpromoter, e.g., a truncated PGK promoter as described herein.

In one embodiment, the vector is an in vitro transcribed vector, e.g., avector that transcribes RNA of a nucleic acid molecule described herein.In one embodiment, the nucleic acid sequence in the vector furthercomprises a poly(A) tail, e.g., a poly A tail described herein, e.g.,comprising about 150 adenosine bases (SEQ ID NO: 377). In oneembodiment, the nucleic acid sequence in the vector further comprises a3′UTR, e.g., a 3′ UTR described herein, e.g., comprising at least onerepeat of a 3′UTR derived from human beta-globulin. In one embodiment,the nucleic acid sequence in the vector further comprises promoter,e.g., a T2A promoter.

In another aspect, the invention pertains to a cell comprising a vectordescribed herein. In one embodiment, the cell is a cell describedherein, e.g., an immune effector cell, e.g., a human T cell, e.g., ahuman T cell described herein, or a human NK cell, e.g., a human NK celldescribed herein. In one embodiment, the human T cell is a CD8+ T cell.

In one embodiment, the CAR-expressing cell described herein can furtherexpress another agent, e.g., an agent which enhances the activity of aCAR-expressing cell. For example, in one embodiment, the agent can be anagent which inhibits an inhibitory molecule. Examples of inhibitorymolecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g.,CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 orCD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFRbeta, e.g., as described herein. In one embodiment, the agent whichinhibits an inhibitory molecule comprises a first polypeptide, e.g., aninhibitory molecule, associated with a second polypeptide that providesa positive signal to the cell, e.g., an intracellular signaling domaindescribed herein. In one embodiment, the agent comprises a firstpolypeptide, e.g., of an inhibitory molecule such as PD1, PD-L1, PD-L2,LAG3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), CTLA4, VISTA,CD160, BTLA, LAIR1, TIM3, 2B4, TGFR beta, CD80, CD86, B7-H3 (CD276),B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHCclass II, GAL9, adenosine, and TIGIT, or a fragment of any of these(e.g., at least a portion of the extracellular domain of any of these),and a second polypeptide which is an intracellular signaling domaindescribed herein (e.g., comprising a costimulatory domain (e.g., 41BB,CD27 or CD28, e.g., as described herein) and/or a primary signalingdomain (e.g., a CD3 zeta signaling domain described herein). In oneembodiment, the agent comprises a first polypeptide of PD1 or a fragmentthereof (e.g., at least a portion of the extracellular domain of PD1),and a second polypeptide of an intracellular signaling domain describedherein (e.g., a CD28 signaling domain described herein and/or a CD3 zetasignaling domain described herein).

In another aspect, the invention pertains to a method of making a cellcomprising transducing a cell described herein, e.g., an immune effectorcell described herein, e.g., a T cell described herein or an NK cell,with a vector of comprising a nucleic acid encoding a CAR, e.g., a CARdescribed herein.

The present invention also provides a method of generating a populationof RNA-engineered cells, e.g., cells described herein, e.g., immuneeffector cells, e.g., T cells or NK cells, transiently expressingexogenous RNA. The method comprises introducing an in vitro transcribedRNA or synthetic RNA into a cell, where the RNA comprises a nucleic acidencoding a CAR molecule described herein.

In another aspect, the invention pertains to a method of providing ananti-tumor immunity in a mammal comprising administering to the mammalan effective amount of a cell expressing a CAR molecule, e.g., a cellexpressing a CAR molecule described herein. In one embodiment, the cellis an autologous immune effector cell, e.g., T cell or NK cell. In oneembodiment, the cell is an allogeneic immune effector cell, e.g., T cellor NK cell. In one embodiment, the mammal is a human, e.g., a patientwith a hematologic cancer.

In another aspect, the invention pertains to a method of treating amammal having a disease associated with expression of CD33 (e.g., aproliferative disease, a precancerous condition, and a noncancer relatedindication associated with the expression of CD33) comprisingadministering to the mammal an effective amount of the cells expressinga CAR molecule, e.g., a CAR molecule described herein. In oneembodiment, the mammal is a human, e.g., a patient with a hematologiccancer.

In one embodiment, the disease is a disease described herein. In oneembodiment, the disease associated with CD33 expression is selected froma proliferative disease such as a cancer or malignancy or a precancerouscondition such as a myelodysplasia, a myelodysplastic syndrome or apreleukemia, or is a non-cancer related indication associated withexpression of CD33. In one embodiment, the disease is a hematologiccancer selected from the group consisting of one or more acute leukemiasincluding but not limited to acute myeloid leukemia (AML);myelodysplastic syndrome; myeloproliferative neoplasms; chronic myeloidleukemia (CML); Blastic plasmacytoid dendritic cell neoplasm; and todisease associated with CD33 expression including, but not limited toatypical and/or non-classical cancers, malignancies, precancerousconditions or proliferative diseases expressing CD33; and combinationsthereof. In one embodiment, the cells expressing a CAR molecule, e.g., aCAR molecule described herein, are administered in combination with anagent that increases the efficacy of a cell expressing a CAR molecule,e.g., an agent described herein.

In another aspect, the invention pertains to a method of conditioning asubject prior to cell transplantation comprising administering to thesubject an effective amount of the cell of comprising a CAR moleculedisclosed herein. In one embodiment, the cell transplantation is a stemcell transplantation. The stem cell transplantation is a hematopoieticstem cell transplantation or a bone marrow transplantation. In oneembodiment, the cell transplantation is allogeneic or autologous.

In one embodiment, the conditioning a subject prior to celltransplantation comprises reducing the number of CD33-expressing cellsin a subject. The CD33-expressing cells in the subject areCD33-expressing normal cells or CD33-expressing cancer cells, and insome cases, the condition in the subject will reduce bothCD33-expressing normal and cancer cells prior to a cell transplantation.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with a low,immune enhancing dose of an mTOR inhibitor. While not wishing to bebound by theory, it is believed that treatment with a low, immuneenhancing, dose (e.g., a dose that is insufficient to completelysuppress the immune system but sufficient to improve immune function) isaccompanied by a decrease in PD-1 positive immune effector cells, e.g.,T cells or NK cells, or an increase in PD-1 negative cells. PD-1positive immune effector cells (e.g., T cells or NK cells), but not PD-1negative immune effector cells (e.g., T cells or NK cells), can beexhausted by engagement with cells which express a PD-1 ligand, e.g.,PD-L1 or PD-L2.

In an embodiment, this approach can be used to optimize the performanceof CAR cells described herein in the subject. While not wishing to bebound by theory, it is believed that, in an embodiment, the performanceof endogenous, non-modified immune effector cells, e.g., T cells or NKcells, is improved. While not wishing to be bound by theory, it isbelieved that, in an embodiment, the performance of of a CD33 CARexpressing cell is improved. In other embodiments, cells, e.g., immuneeffector cells (e.g., T cells or NK cells), which have, or will beengineered to express a CAR, can be treated ex vivo by contact with anamount of an mTOR inhibitor that increases the number of PD1 negativeimmune effector cells, e.g., T cells NK cells, or increases the ratio ofPD1 negative immune effector cells, e.g., T cells or NK cells/PD1positive immune effector cells, e.g., T cells or NK cells.

In an embodiment, administration of a low, immune enhancing, dose of anmTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or acatalytic inhibitor, is initiated prior to administration of an CARexpressing cell described herein, e.g., immune effector cells (e.g., Tcells or NK cells). In an embodiment, the CAR cells are administeredafter a sufficient time, or sufficient dosing, of an mTOR inhibitor,such that the level of PD1 negative immune effector cells, e.g., T cellsor NK cells, or the ratio of PD1 negative immune effector cells, e.g., Tcells or NK cells/PD1 positive immune effector cells, e.g., T cells orNK cells, has been, at least transiently, increased. In an embodiment,the cell, e.g., immune effector cell (e.g., T cell or NK cell), to beengineered to express a CAR, is harvested after a sufficient time, orafter sufficient dosing of the low, immune enhancing, dose of an mTORinhibitor, such that the level of PD1 negative immune effector cells,e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g.,T cells or NK cells/PD1 positive immune effector cells, e.g., T cells orNK cells, in the subject or harvested from the subject has been, atleast transiently, increased.

In an embodiment, the invention provides an mTOR inhibitor for use inthe treatment of a subject, wherein said mTOR inhibitor enhances animmune response of said subject, and wherein said subject has received,is receiving or is about to receive an immune effector cell thatexpresses a CD33 CAR as described herein.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with an agentthat ameliorates one or more side effect associated with administrationof a cell expressing a CAR molecule, e.g., an agent described herein.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with an agentthat treats the disease associated with CD33, e.g., an agent describedherein. In certain embodiments, the disease associated with CD33 is aproliferative disease such as a cancer or malignancy or a precancerouscondition such as a myelodysplasia, a myelodysplastic syndrome or apreleukemia, or is a non-cancer related indication associated withexpression of CD33.

In certain embodiments, the disease associated with CD33 is ahematologic cancer selected from the group consisting of one or moreacute leukemias including but not limited to acute myeloid leukemia(AML); myelodysplastic syndrome; myeloproliferative neoplasms; chronicmyeloid leukemia (CIVIL); Blastic plasmacytoid dendritic cell neoplasm;and to disease associated with CD33 expression including, but notlimited to atypical and/or non-classical cancers, malignancies,precancerous conditions or proliferative diseases expressing CD33; andcombinations thereof.

In some embodiments, a CD33 CAR described herein targets aCD33-expressing cell. In embodiments, the CD33 CAR described hereintargets an MDS blast. In some embodiments, the MDS blast comprises a 5qdeletion (del(5q)). In embodiments, a CD33 CAR-expressing cell describedherein is used to treat a subject having MDS. In embodiments, a CD33CAR-expressing cell described herein is used to treat a subject havingMDS associated with isolated del(5q).

In embodiments, a CD33 CAR described herein targets a MDSC, e.g., a MDSCin a subject having a cancer (e.g., multiple myeloma, chroniclymphocytic leukemia, or solid malignancies such as ovarian cancer,colon cancer, or breast cancer). In embodiments, the MDSC is lineagenegative (LIN−), HLA-DR negative, and CD33 positive. In someembodiments, a CD33 CAR-expressing cell described herein targets a MDSblast and a MDSC. In embodiments, a CD33 CAR-expressing cell describedherein is used to treat multiple myeloma, chronic lymphocytic leukemia(CLL), or solid malignancies such as ovarian cancer, colon cancer, orbreast cancer.

In another aspect, the invention pertains to the isolated nucleic acidmolecule encoding a CAR of the invention, the isolated polypeptidemolecule of a CAR of the invention, the vector comprising a CAR of theinvention, and the cell comprising a CAR of the invention for use as amedicament, e.g., as described herein.

In another aspect, the invention pertains to a the isolated nucleic acidmolecule encoding a CAR of the invention, the isolated polypeptidemolecule of a CAR of the invention, the vector comprising a CAR of theinvention, and the cell comprising a CAR of the invention for use in thetreatment of a disease expressing CD33, e.g., a disease expressing CD33as described herein.

Additional features and embodiments of the aforesaid compositions andmethods include one or more of the following:

In certain embodiments, the CD33 CAR molecule (e.g., a CD33 CAR nucleicacid or a CD33 CAR polypeptide as described herein), or the CD33 bindingdomain as described herein, includes one, two or three CDRs from theheavy chain variable region (e.g., HC CDR1, HC CDR2 and/or HC CDR3),provided in Table 3; and/or one, two or three CDRs from the light chainvariable region (e.g., LC CDR1, LC CDR2 and/or LC CDR3) of CAR33-1,CAR33-2, CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9,provided in Table 4; or a sequence substantially identical (e.g., 95-99%identical, or up to 5, 4, 3, 2, or 1 amino acid changes, e.g.,substitutions (e.g., conservative substitutions)) to any of theaforesaid sequences.

In certain embodiments, the CD33 CAR molecule (e.g., a CD33 CAR nucleicacid or a CD33 CAR polypeptide as described herein), or the anti-CD33antigen binding domain as described herein, includes one, two or threeCDRs from the heavy chain variable region (e.g., HC CDR1, HC CDR2 and/orHC CDR3), provided in Table 10; and/or one, two or three CDRs from thelight chain variable region (e.g., LC CDR1, LC CDR2 and/or LC CDR3) ofCAR33-1, CAR33-2, CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8,CAR33-9, provided in Table 11; or a sequence substantially identical(e.g., 95-99% identical, or up to 5, 4, 3, 2, or 1 amino acid changes,e.g., substitutions (e.g., conservative substitutions)) to any of theaforesaid sequences.

In certain embodiments, the CD33 CAR molecule, or the anti-CD33 antigenbinding domain, includes one, two or three CDRs from the heavy chainvariable region (e.g., HCDR1, HCDR2 and/or HCDR3), provided in Table 12;and/or one, two or three CDRs from the light chain variable region(e.g., LC CDR1, LC CDR2 and/or LC CDR3) of CAR33-1, CAR33-2, CAR33-3,CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9, provided in Table13; or a sequence substantially identical (e.g., 95-99% identical, or upto 5, 4, 3, 2, or 1 amino acid changes, e.g., substitutions (e.g.,conservative substitutions)) to any of the aforesaid sequences.

In certain embodiments, the CD33 CAR molecule, or the anti-CD33 antigenbinding domain, includes

(i) a LC CDR1, LC CDR2 and LC CDR3 of any CD33 light chain bindingdomain amino acid sequences listed in Table 2 or 9, or the LC CDRs inTable 4, 9, 11 or 13; and/or.

(ii) a HC CDR1, HC CDR2 and HC CDR3 of any CD33 heavy chain bindingdomain amino acid sequences listed in Table 2 or 9, or the HC CDRs inTable 3, 9, 10 or 12.

In certain embodiments, the CD33 CAR molecule (e.g., a CD33 CAR nucleicacid or a CD33 CAR polypeptide as described herein), or the anti-CD33antigen binding domain as described herein, includes:

(1) three light chain (LC) CDRs chosen from one of the following:

(i) a LC CDR1 of SEQ ID NO: 111, LC CDR2 of SEQ ID NO: 120 and LC CDR3of SEQ ID NO: 129 of CAR33-1;

(ii) a LC CDR1 of SEQ ID NO: 112, LC CDR2 of SEQ ID NO: 121 and LC CDR3of SEQ ID NO: 130 of CAR33-2;

(iii) a LC CDR1 of SEQ ID NO: 113, LC CDR2 of SEQ ID NO: 122 and LC CDR3of SEQ ID NO: 131 of CAR33-3;

(iv) a LC CDR1 of SEQ ID NO: 114, LC CDR2 of SEQ ID NO: 123 and LC CDR3of SEQ ID NO: 132 of CAR33-4;

(iv) a LC CDR1 of SEQ ID NO: 115, LC CDR2 of SEQ ID NO: 124 and LC CDR3of SEQ ID NO: 133 of CAR33-5;

(vi) a LC CDR1 of SEQ ID NO: 116, LC CDR2 of SEQ ID NO: 125 and LC CDR3of SEQ ID NO: 134 of CAR33-6;

(vii) a LC CDR1 of SEQ ID NO: 117, LC CDR2 of SEQ ID NO: 126 and LC CDR3of SEQ ID NO: 135 of CAR33-7;

(viii) a LC CDR1 of SEQ ID NO: 118, LC CDR2 of SEQ ID NO: 127 and LCCDR3 of SEQ ID NO: 136 of CAR33-8; or

(ix) a LC CDR1 of SEQ ID NO: 119, LC CDR2 of SEQ ID NO: 128 and LC CDR3of SEQ ID NO: 137 of CAR33-9; and/or

(2) three heavy chain (HC) CDRs chosen from one of the following:

(i) a HC CDR1 of SEQ ID NO: 84, HC CDR2 of SEQ ID NO: 93 and HC CDR3 ofSEQ ID NO: 102 of CAR33-1;

(ii) a HC CDR1 of SEQ ID NO: 85, HC CDR2 of SEQ ID NO: 94 and HC CDR3 ofSEQ ID NO: 103 of CAR33-2;

(iii) a HC CDR1 of SEQ ID NO: 86, HC CDR2 of SEQ ID NO: 95 and HC CDR3of SEQ ID NO: 104 of CAR33-3;

(iv) a HC CDR1 of SEQ ID NO: 87, HC CDR2 of SEQ ID NO: 96 and HC CDR3 ofSEQ ID NO: 105 of CAR33-4;

(iv) a HC CDR1 of SEQ ID NO: 88, HC CDR2 of SEQ ID NO: 97 and HC CDR3 ofSEQ ID NO: 106 of CAR33-5;

(vi) a HC CDR1 of SEQ ID NO: 89, HC CDR2 of SEQ ID NO: 98 and HC CDR3 ofSEQ ID NO: 107 of CAR33-6;

(vii) a HC CDR1 of SEQ ID NO: 90, HC CDR2 of SEQ ID NO: 99 and HC CDR3of SEQ ID NO: 108 of CAR33-7;

(viii) a HC CDR1 of SEQ ID NO: 91, HC CDR2 of SEQ ID NO: 100 and HC CDR3of SEQ ID NO: 109 of CAR33-8; or

(ix) a HC CDR1 of SEQ ID NO: 92, HC CDR2 of SEQ ID NO: 101 and HC CDR3of SEQ ID NO: 110 of CAR33-9.

In certain embodiments, the CD33 CAR molecule (e.g., a CD33 CAR nucleicacid or a CD33 CAR polypeptide as described herein), or the anti-CD33antigen binding domain as described herein, includes:

(1) three light chain (LC) CDRs chosen from one of the following:

(i) a LC CDR1 of SEQ ID NO: 296, LC CDR2 of SEQ ID NO: 305 and LC CDR3of SEQ ID NO: 314 of CAR33-1;

(ii) a LC CDR1 of SEQ ID NO: 297, LC CDR2 of SEQ ID NO: 306 and LC CDR3of SEQ ID NO: 315 of CAR33-2;

(iii) a LC CDR1 of SEQ ID NO: 298, LC CDR2 of SEQ ID NO: 307 and LC CDR3of SEQ ID NO: 316 of CAR33-3;

(iv) a LC CDR1 of SEQ ID NO: 299, LC CDR2 of SEQ ID NO: 308 and LC CDR3of SEQ ID NO: 317 of CAR33-4;

(iv) a LC CDR1 of SEQ ID NO: 300, LC CDR2 of SEQ ID NO: 309 and LC CDR3of SEQ ID NO: 318 of CAR33-5;

(vi) a LC CDR1 of SEQ ID NO: 301, LC CDR2 of SEQ ID NO: 310 and LC CDR3of SEQ ID NO: 319 of CAR33-6;

(vii) a LC CDR1 of SEQ ID NO: 302, LC CDR2 of SEQ ID NO: 311 and LC CDR3of SEQ ID NO: 320 of CAR33-7;

(viii) a LC CDR1 of SEQ ID NO: 303, LC CDR2 of SEQ ID NO: 312 and LCCDR3 of SEQ ID NO: 321 of CAR33-8; or

(ix) a LC CDR1 of SEQ ID NO: 304, LC CDR2 of SEQ ID NO: 313 and LC CDR3of SEQ ID NO: 322 of CAR33-9; and/or

(2) three heavy chain (HC) CDRs chosen from one of the following:

(i) a HC CDR1 of SEQ ID NO: 269, HC CDR2 of SEQ ID NO: 278 and HC CDR3of SEQ ID NO: 287 of CAR33-1;

(ii) a HC CDR1 of SEQ ID NO: 270, HC CDR2 of SEQ ID NO: 279 and HC CDR3of SEQ ID NO: 288 of CAR33-2;

(iii) a HC CDR1 of SEQ ID NO: 271, HC CDR2 of SEQ ID NO: 280 and HC CDR3of SEQ ID NO: 289 of CAR33-3;

(iv) a HC CDR1 of SEQ ID NO: 272, HC CDR2 of SEQ ID NO: 281 and HC CDR3of SEQ ID NO: 290 of CAR33-4;

(iv) a HC CDR1 of SEQ ID NO: 273, HC CDR2 of SEQ ID NO: 282 and HC CDR3of SEQ ID NO: 291 of CAR33-5;

(vi) a HC CDR1 of SEQ ID NO: 274, HC CDR2 of SEQ ID NO: 283 and HC CDR3of SEQ ID NO: 292 of CAR33-6;

(vii) a HC CDR1 of SEQ ID NO: 275, HC CDR2 of SEQ ID NO: 284 and HC CDR3of SEQ ID NO: 293 of CAR33-7;

(viii) a HC CDR1 of SEQ ID NO: 276, HC CDR2 of SEQ ID NO: 285 and HCCDR3 of SEQ ID NO: 294 of CAR33-8; or

(ix) a HC CDR1 of SEQ ID NO: 277, HC CDR2 of SEQ ID NO: 286 and HC CDR3of SEQ ID NO: 295 of CAR33-9.

In certain embodiments, the CD33 CAR molecule (e.g., a CD33 CAR nucleicacid or a CD33 CAR polypeptide as described herein), or the anti-CD33antigen binding domain as described herein, includes:

(1) three light chain (LC) CDRs chosen from one of the following:

(i) a LC CDR1 of SEQ ID NO: 350, LC CDR2 of SEQ ID NO: 359 and LC CDR3of SEQ ID NO: 368 of CAR33-1;

(ii) a LC CDR1 of SEQ ID NO: 351, LC CDR2 of SEQ ID NO: 360 and LC CDR3of SEQ ID NO: 369 of CAR33-2;

(iii) a LC CDR1 of SEQ ID NO: 352, LC CDR2 of SEQ ID NO: 361 and LC CDR3of SEQ ID NO: 370 of CAR33-3;

(iv) a LC CDR1 of SEQ ID NO: 353, LC CDR2 of SEQ ID NO: 362 and LC CDR3of SEQ ID NO: 371 of CAR33-4;

(iv) a LC CDR1 of SEQ ID NO: 354, LC CDR2 of SEQ ID NO: 363 and LC CDR3of SEQ ID NO: 372 of CAR33-5;

(vi) a LC CDR1 of SEQ ID NO: 355, LC CDR2 of SEQ ID NO: 364 and LC CDR3of SEQ ID NO: 373 of CAR33-6;

(vii) a LC CDR1 of SEQ ID NO: 356, LC CDR2 of SEQ ID NO: 365 and LC CDR3of SEQ ID NO: 374 of CAR33-7;

(viii) a LC CDR1 of SEQ ID NO: 357, LC CDR2 of SEQ ID NO: 366 and LCCDR3 of SEQ ID NO: 375 of CAR33-8; or

(ix) a LC CDR1 of SEQ ID NO: 358, LC CDR2 of SEQ ID NO: 367 and LC CDR3of SEQ ID NO: 376 of CAR33-9; and/or

(2) three heavy chain (HC) CDRs chosen from one of the following:

(i) a HC CDR1 of SEQ ID NO: 323, HC CDR2 of SEQ ID NO: 332 and HC CDR3of SEQ ID NO: 341 of CAR33-1;

(ii) a HC CDR1 of SEQ ID NO: 324, HC CDR2 of SEQ ID NO: 333 and HC CDR3of SEQ ID NO: 342 of CAR33-2;

(iii) a HC CDR1 of SEQ ID NO: 325, HC CDR2 of SEQ ID NO: 334 and HC CDR3of SEQ ID NO: 343 of CAR33-3;

(iv) a HC CDR1 of SEQ ID NO: 326, HC CDR2 of SEQ ID NO: 335 and HC CDR3of SEQ ID NO: 344 of CAR33-4;

(iv) a HC CDR1 of SEQ ID NO: 327, HC CDR2 of SEQ ID NO: 336 and HC CDR3of SEQ ID NO: 345 of CAR33-5;

(vi) a HC CDR1 of SEQ ID NO: 328, HC CDR2 of SEQ ID NO: 337 and HC CDR3of SEQ ID NO: 346 of CAR33-6;

(vii) a HC CDR1 of SEQ ID NO: 329, HC CDR2 of SEQ ID NO: 338 and HC CDR3of SEQ ID NO: 347 of CAR33-7;

(viii) a HC CDR1 of SEQ ID NO: 330, HC CDR2 of SEQ ID NO: 339 and HCCDR3 of SEQ ID NO: 348 of CAR33-8; or

(ix) a HC CDR1 of SEQ ID NO: 331, HC CDR2 of SEQ ID NO: 340 and HC CDR3of SEQ ID NO: 349 of CAR33-9.

In certain embodiments, the CD33 CAR molecule (e.g., a CD33 CAR nucleicacid or a CD33 CAR polypeptide as described herein), or the anti-CD33antigen binding domain as described herein, includes the 2213 scFv aminoacid sequence (SEQ ID NO: 142) or a nucleotide sequence encoding the2213 scFv (SEQ ID NO: 141), or an antigen binding domain thereof (e.g.,a VH, VL or one or more CDRs thereof).

In certain embodiments, the CD33 CAR molecule (e.g., a CD33 CAR nucleicacid or a CD33 CAR polypeptide as described herein), or the anti-CD33antigen binding domain as described herein, includes the my96 scFv aminoacid sequence (SEQ ID NO: 147), or an antigen binding domain thereof(e.g., a VH, VL or one or more CDRs thereof).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.Headings, sub-headings or numbered or lettered elements, e.g., (a), (b),(i) etc., are presented merely for ease of reading. The use of headingsor numbered or lettered elements in this document does not require thesteps or elements be performed in alphabetical order or that the stepsor elements are necessarily discrete from one another. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 is an image demonstrating CD33 is expressed on most blasts inmany primary patient samples with AML (AML blasts were gated usingstandard side scatter ^(low) CD45^(dim) characteristics); n=35-46 pergroup.

FIGS. 2A, 2B, and 2C are graphs and a flow cytometry profile showing theexpression of CD33 in bone marrow from myelodysplastic syndromepatients. FIG. 2A shows the percentage of CD33-expressing cells in theCD34+CD38− hematopoietic stem cell compartment in MDS patients. FIG. 2Bshows the percentage of CD33-expressing cells in the CD34+CD38+compartment containing myeloid progenitors in MDS patients. FIG. 2C is ahistogram showing the mean fluorescence intensity from an MDS patient inthe CD34+CD38− compartment.

FIG. 3 shows a schematic representation of CAR constructs used inExample 1. All are second generation CARs using 41BB and CD3zetasignaling. The scFv of CART33 was derived from clone MY9-6.

FIG. 4A is a set of images and FIG. 4B is a graph demonstrating in vitroactivity of CART33. CART33-mediated T cell degranulation:CAR33-transduced and untransduced T cells were incubated with the CD33+cell line MOLM14 and a control ALL cell line NALM6 for 4 hours in thepresence of CD28, CD49d and monensin. CD107a degranulation was measuredby flow cytometry. Expression of both murine and humanized CART33constructs elicit specific degranulation in the presence of MOLM14(P<0.001).

FIG. 5A is a set of images and FIG. 5B is a graph demonstrating in vitroactivity of CART33. Cytokine production: both humanized and murine CAR33expressing T cells produce cytokine after incubation with MOLM14. Tcells were incubated with the CD33+ cell line MOLM14 and a control cellline NALM6 for 4 hours. Cells were then fixed, permeabilized and stainedfor intracellular tumor necrosis alpha and interferon gamma. Sampleswere then alayzed by by flow cytometry.

FIG. 6 is an image demonstrating in vitro activity of CART33.Proliferation of CAR123- and CAR33-expressing T cells: humanized CART33and murine CART33 proliferation in response to MOLM14. T cells werelabeled with CFSE and incubated under control conditions or with MOLM14for 120 hours and CFSE dilution was measured by flow cytometry as amarker of proliferation.

FIGS. 7A and 7B are two graphs demonstrating in vitro activity ofCART33. Specific killing of CAR123-, humanized CAR33-, and murineCAR33-expressing T cells: T cells were incubated with MOLM14 or theT-cell ALL cell line Jurkat (control) for 24 hours. huCART33 resulted insignificantly more specific killing compared to murine CART33 at low E:Tratios. In FIG. 7B, CART33-Jurkat is represented by triangles,CART33-MOLM14 is represented by upside down triangles, andCART123-MOLM14 is represented by squares.

FIG. 8 is an image demonstrating CART33 (IgG4 hinge) and CART33 (CD8hinge) have equivalent in vitro activity. Degranulation assay: CART33(IgG4 hinge), CART33 (CD8 hinge), CART123, and untransduced T cells wereincubated with the CD33+ cell line MOLM14 and CD107a degranulation wasmeasured by flow cytometry. Both CART33 constructs undergo specificdegranulation in the presence of MOLM14.

FIG. 9 is an image demonstrating CART33 (IgG4 hinge) and CART33 (CD8hinge) have equivalent in vitro activity. Cytokine production: bothCAR33 constructs and CAR123 specifically induce cytokine productionafter incubation with the MOLM14 cell line. T cells were incubated withMOLM14 for 4 hours in the presence of CD28, CD49d and monensin. Cellswere then harvested, fixed, permalized and stained for tumor necrosisalpha, MIP1a and interferon gamma. Percentage of cells producingcytokines were then measured by flow cytometry.

FIG. 10 is an image demonstrating CART33 (IgG4 hinge) and CART33 (CD8hinge) have equivalent in-vitro activity. Proliferation of controluntransduced, CAR33- (IgG4 hinge) (i.e., CAR33-IgG4H), CAR33- (CD8hinge) (i.e., CAR33-CD8H), or CAR123-expressing T cells in response toMOLM14. T cells were labeled with CFSE and incubated with MOLM14 for 120hours.

FIG. 11 is a schematic diagram demonstrating a comparison of in vivoanti-tumor effect of CART33-CD8H, CART33-IgG4H, and CART123.NOD-SCID-common gamma chain knockout (NSG) mice were injected with theAML cell line MOLM14 1×10⁶ i.v. and imaged for engraftment after 6 days.On day 7, mice were treated with T cells expressing CAR33 (IgG4 hinge),CAR33 (CD8 hinge), CAR123, or control vehicle (untransduced cells).Total number of T cells injected was 2×10⁶ i.v. The mice were followedwith serial weekly imaging to assess tumor burden.

FIG. 12 is an image demonstrating equivalent in vivo anti-tumor effectof CART33-CD8H, CART33-IgG4H, and CART123 T cells. Tumor burden overtime by bioluminescent imaging (BLI); data from one experiment (n=5 pergroup), representative of 4 independent experiments of the mice shown inFIG. 11.

FIG. 13 is a schematic diagram demonstrating a comparison of CART33 andCART123 eradication of primary AML in vivo. NSG mice transgenic for thehuman cytokines IL3/GM-CSF/SCF (NSGS mice) were injected with a primaryAML sample at 5×10⁶ i.v. Engraftment was confirmed by retro-orbitalbleeding after 2-4 weeks and then mice were treated with CART33,CART123, or control vehicle (untransduced cells). Total number of Tcells injected was 1×10⁵ i.v. The mice were followed with serialretro-orbital bleedings to assess the burden of AML.

FIGS. 14A, 14B, and 14C are sets of images demonstrating CART33 andCART123 produce equivalent eradication of primary AML in vivo. Analysisof peripheral blood from mice treated with untransduced (UTD), CART33 orCART123 at baseline, day 14 and day +70. AML according to theexperimental set-up described in FIG. 13 was not detected in micetreated with CART33 or CART123.

FIG. 15 is an image demonstrating CART33 and CART123 produce equivalenteradication of primary AML in vivo. Summary of disease burden measuredby blasts/ul from retro-orbital bleedings at different time points asindicated from mice in experimental set-up described in FIG. 13.

FIG. 16 is an image demonstrating CART33 and CART123 produce equivalenteradication of primary AML in vivo. Survival of mice treated withCART33, CART123 or UTD (p<0.001 when treatment with either CART33 orCART123 is compared to UTD) according to the experimental set-updescribed in FIG. 13.

FIG. 17 is a schematic diagram demonstrating a set-up for testinghematopoietic stem cell toxicity of CART33 cells. Schema of theexperiment: humanized immune system (HIS) mice were bled retro-orbitally6-8 weeks after injection of human CD34+ cells derived from the fetalliver to confirm engraftment of human cells. Mice were then treated witheither CART33 or UTD (1×10⁶ cells each) and followed by serial weeklyretro-orbital bleedings. Mice were then euthanized on day 28 and organswere harvested and analyzed.

FIGS. 18A, 18B, and 18C are sets of images demonstrating hematopoieticstem cell toxicity of CART33 cells. Analysis of the peripheral blood(via retro-orbital bleeding) by flow cytometry from day 28 at theconclusion of the experiment shown in FIG. 17. CART33 treatment leads tosignificant reduction in peripheral blood myeloid cells and monocytescompared to treatment with untransduced T cells.

FIG. 19 is an image demonstrating hematopoietic stem cell toxicity ofCART33 cells. Summary statistics of day 28 peripheral blood analysisfrom mice treated with CART33, UTD or no treatment (n=5) from theexperiment shown in FIG. 17. CART33 resulted in significant toxicity onmonocytes and CD33⁺ myeloid lineage cells with relative sparing of Bcells and platelets.

FIG. 20 is an image demonstrating hematopoietic stem cell toxicity ofCART33 cells. Plots from bone marrow analysis by flow cytometry on day28 of mice from the experiment shown in FIG. 17. CART33 treatmentresulted in significant reduction in myeloid progenitors (CD34+CD38+)and in hematopoietic stem cells (CD34+CD38-), gated on singlets,huCD45dim, Lineage negative.

FIG. 21 is an image demonstrating hematopoietic stem cell toxicity ofCART33 cells. Sections of the femur of mice from the experiment shown inFIG. 17 were taken from the mice on day 28 after treatment with UTD Tcells or CART33 cells. huCD45 and CD34 staining by IHC was performed. Nodifference in huCD45 between control T cells and CART33, although boththese groups show less huCD45 staining likely consistent with anallogeneic human-anti-human effect. There was specific reduction ofCD34+ cells in mice treated with CART33. Results are representative oftwo experiments.

FIG. 22 is a schematic diagram demonstrating a set-up for testing CART33and CART123 hematopoietic toxicity in vivo. Schema of the experiment:NSGS mice received busulfan i.p. followed by 2×10⁶ T cell depleted bonemarrow cells from a normal donor the following day. Engraftment wasconfirmed by flow cytometric analysis of peripheral blood after 4 weeksand mice were then treated with 1×10⁶ autologous T cells, transducedwith CART33, CART123 or UTD. Mice were then followed with retro-orbitalbleeding on day 7 and day 14 and were euthanized for necropsy on day 14.

FIG. 23 is an image demonstrating CART33 and CART123 produce equivalenthematopoietic toxicity in vivo. Shown is a representative plot of bonemarrow analysis from mice from the experiment shown in FIG. 22 by flowcytometry on day 28. CART33 and CART123 treatment resulted insignificant reduction in myeloid progenitors (CD34+CD38+) and inhematopoietic stem cells (CD34+CD38−), gated on huCD45dim, Lin−. Resultsare representative of two experiments.

FIG. 24 is an image demonstrating CART33 and CART123 are cytotoxicmyelodysplastic syndrome (MDS) marrow cells. CD34 enriched BM frompatients with MDS was incubated with either UTD, CART33 (IgG4 hinge),CART33 (CD8 hinge), or CART123. There was reduction in CD45dimCD34+cells in samples treated with CART33 or CART123.

FIG. 25 is a schematic diagram of a vector for expressing a murineCART33.

FIG. 26 is a schematic diagram of a vector for expressing a humanizedCART33.

FIG. 27 is an image depicting the cell surface expression of scFvs on aJurkat T cell line, which contains a luciferase reporter driven by anNFAT-regulated promoter (termed JNL cells). JNL cells were transducedwith a lentiviral vector expressing a cDNA encoding GFP, a scFv thatbinds to CD19, or cDNAs that encode an scFv, which was raised againsthsCD33. The cell surface expression of individual scFv's on JNLs wasdetected by incubating cells with recombinant Fc-tagged hsCD33 followedby incubation with an Fc-specific secondary antibody conjugated tophycoerythrin.

FIGS. 28A, 28B, 28C, and 28D are images showing the ability ofindividual scFv's targeting hsCD33 to elicit NFAT activity in JNL cells.JNL cells expressing scFv's against hsCD33 were co-cultured with MOLM13or MOLP8 cell lines, which express hsCD33 or lack hsCD33 cell surfaceexpression, respectively (FIG. 28A; hsCD33, solid green line; isotypecontrol, gray dashed line and shaded area). FIG. 28B depicts theactivation of JNL cells expressing an scFv targeting hsCD33 in thepresence of MOLM13 (solid lines) or MOLP8 (dashed lines) cells. JNLcells expressing individual scFv's were plated at different effector(i.e. JNL cells) to target (i.e. MOLM13 or MOLP8) ratios and analyzedfor the expression of relative luciferase units (RLUs) using theBright-Glo™ Luciferase Assay on the EnVision instrument 24 hourspost-incubation. FIG. 28C is a version of FIG. 28B, depicting theactivation of JNL cells in the presence of MOLM13. FIG. 28D is a versionof FIG. 28B, depicting the activation of JNL cells in the presence ofMOLP8.

FIGS. 29A and 29B are panels of images depicting the activity of scFv'stargeting hsCD33 in donor-derived primary T cells. Expression of scFv'son the cell surface of primary human T cells transduced with alentiviral vector that expresses an scFv that targets hsCD33 isdepicted. The expression of scFv's was detected by incubating cells withrecombinant Fc-tagged hsCD33 and an Fc-specific secondary antibodyconjugated to phycoerythrin as described in FIG. 27.

FIGS. 30A and 30B are images depicting the proliferative activity of Tcells expressing scFvs targeting CD33. T cells were labeled with CFSEand co-cultured in the presence of MOLM13 (FIG. 30A, solid black bar),MOLP8 (FIG. 30A, open white bar), or cultured alone (FIG. 30A, hatchedbar) to assess the proliferative capacity of UTD primary T cells orcells expressing scFvs targeting hsCD33. In addition, antigen drivencell division in primary T cells was assessed by measuring the medianfluorescence intensity (MFI) of CF SE-labeled T cells expressing scFvclones CD33-2, -3, -4, -5, -6, and -9 co-cultured with MOLM13, MOLP8cells, or alone (FIG. 30B).

FIG. 31 is an image depicting cytolytic activity of T cells expressingscFVs targeting CD33. To assess cytolytic activity, 25,000 MOLM13 cellswere plated with primary T cells expressing individual scFv's atdifferent effector (i.e. T cell) to target (i.e. MOLM13) ratios andanalyzed for the extent of MOLM13 killing by enumerating the absolutenumber of CFSE-labeled MOLM13 cells after 4 days in culture.

FIG. 32 is an image depicting cross-reactivity of T cells expressingscFvs targeting CD33 to cynomolgus CD33 (cyCD33) by flow cytometry. JNLcells transduced with a lentiviral vector expressing scFv's raisedagainst hsCD33 were incubated with either recombinant Fc-tagged hsCD33(dotted line) or cyCD33 (solid line) followed by incubation with anFc-specific secondary antibody conjugated to phycoerythrin.

FIGS. 33A and 33B depict the expression of an mRNA CAR33 in T cells fromnormal donors after electroporation. FIG. 33A is a series of flowcytometry profiles showing the expression of CAR33 in the T cellpopulation at the indicated time points. The percentage ofCAR33-expressing cells (boxed) are quantified and shown in the profile.FIG. 33B is a graphic representation of the percentage of CAR33expression.

FIGS. 34A and 34B compare the expression of lentivirally-transducedCAR33 to mRNA-electroporated CAR33. FIG. 34A shows the stable expressionof lentivirally-transduced CAR33 (CART33LV) at the indicated times, over4 days. FIG. 34B shows the transient expression of mRNA-electroporatedCAR33 (CART33 RNA) at the indicated times, over 4 days. The expressionof CAR33 is represented by mean fluorescence intensity (MFI) on thex-axis, and total cell number is represented on the y-axis.

FIGS. 35A, 35B, 35C, and 35D are graphic representations comparing thecytolytic activity of T cells expressing CD33 after lentiviraltransduction or mRNA electroporation. The experiment was repeated at 1day (FIG. 35A), 2 days (FIG. 35B), 3 days (FIG. 35C), and 4 days (FIG.35D) post electroporation of the T cells. CART33 cells were incubatedwith the CD33 positive cell line MOLM14 and a control mantle celllymphoma cell line JEKO at the E:T (effector to target) ratios indicatedin the x-axis. Percentage killing at each ratio is indicated in they-axis.

FIGS. 36A and 36B are graphic representations comparing the cytolyticactivity of T cells expressing CD33 after lentiviral transduction ormRNA electroporation over time. FIG. 36A shows the specific killing oflentivirally-transduced CAR33 cells compared to RNA CAR33 cells whenincubated with MOLM14 cells at the E:T (effector:target) ratio of 2:1over 4 days. FIG. 36B shows the specific killing oflentivirally-transduced CAR33 cells compared to RNA CAR33 cells whenincubated with MOLM14 cells at the E:T (effector:target) ratio of 1:1over 4 days.

FIGS. 37A, 37B, 37C, and 37D show that CART33 cells exhibit robust invitro effector functions in response to the CD33+ cell line MOLM14 or toprimary AML samples. Plots are representative of four independentexperiments. FIG. 37A shows the CD107a degranulation. CART33, CART123and untransduced T cells (UTD) were incubated with the CD33+/CD123+ cellline MOLM14, PMA/Ionomycin as positive non specific T cell stimulant andthe control T cell ALL cell line Jurkat, in the presence of CD49d, CD28costimulatory molecules and momensin. CD107a degranulation was measuredby flow cytometry after 4 hours of incubation. FIG. 37B shows thespecific killing of CD33-expressing cells. CART33, CART123 and UTD wereincubated with MOLM14-luc or Jurkat-luc for 24 hours at different E:Tratios as indicated and bioluminescence imaging was then performed as ameasure of residual living cells. The black/solid line (squares)represents CART123 incubated with MOLM14; the blue/dotted line(triangles pointing down/filled in triangles) represents CART33incubated with MOLM14; and the red/dashed line (triangles pointingup/open triangles) represents CART33 incubated with Jurkat. FIGS. 37Cand 37D show the proliferation of CART33 cells in response toCD33-expressing cells. T cells were labeled with CFSE and incubated withMOLM14, PMA/IONO as positive non specific T cell stimulant, Jurkat as anegative control, or AML samples for 120 hours. The number ofproliferating T cells was significantly higher in response to MOLM14 ascompared to Jurkat and was comparable to CART123.

FIGS. 38A, 38B, and 38C show the cytokine production by CART33 cells inresponse to CD33-expressing cells MOLM14. CART33, CART123 and UTD cellswere incubated with MOLM14, PMA/Ionomycin, and Jukat for 4 hours. Thecells were then fixed and permeabilized, stained for 5 differentcytokines (Tumor necrosis factor alpha, interferon gamma, granulocytemacrophage colony stimulating factor, macrophase inflammatory protein1b, and interleukin-2), and flow cytometric analyses were performed. Themajority of CAR T33 cells produce more than one cytokine in response toMOLM14 (FIG. 38A), similar to their response to PMA/Ionomycin (positivecontrol, FIG. 38B). FIG. 38C shows the production of IL-2, IFN-γ,GM-CSF, and TNF-α in response to MOLM14 was significantly higher inCART33 than CART123 cells. CART33, CART123 and UTD cells were incubatedwith MOLM14, Jurkat and PMA/Ionomycin for 24 hours. Supernatant was thenharvested and a 30-plex Luminex assay was performed. Levels of the restof cytokines are presented in FIGS. 39A-39C.

FIGS. 39A, 39B, and 39C are a series of graphs showing the comparison ofcytokine production by CART33 and CART123 cells in response to MOLM14.CART33, CART123 and UTD cells were incubated with MOLM14, Jurkat andPMA/Ionomycin for 24 hours. Supernatant was then harvested and a 30-plexLuminex assay was performed for the indicated cytokines.

FIG. 40 shows the specific killing of CD33-expressing MOLM14 cells invitro. CART123, CART33 (CD8 hinge) and CART33 (IgG4 hinge) wereincubated with MOLM14 at the indicated E:T ratios and killing wasassessed by bioluminescence imagine. CART33 (IgG4 hinge) resulted inmore specific killing than CART33 (CD8 hinge) at lower E:T ratio.

FIGS. 41A, 41B, and 41C show the anti-tumor activity in myelodysplasticsyndromes (MDS). FIG. 41A is a graph showing specific CD107adegranulation in response to bone marrow cells from MDS patients. FIG.41B is a set of images showing specific killing of the MDS clone having5q deletion. FIG. 41C is a graph showing the quantification of 5qdeletion clones remaining after treatment as determined by FISH. Therewas significant reduction in the 5q− clone percentage in the grouptreated with CART33 when compared to UTD and No treatment groups.

FIGS. 42A, 42B, 42C show the CART33 treatment and survival results fromMOLM14 engrafted xenografts. The experimental schema is presented inFIG. 11. In FIG. 42A, tumor burden over time by bioluminescent imaging(BLI) was quantified; data from one experiment (n=5 per group), eachmouse is represented by a line. FIG. 42B shows the composite survival ofthree independent experiments. Treatment with CART33 resulted insignificant survival advantages when compared with treatment with UTD.FIG. 42C are representative bioluminescence images from one experiment.

FIGS. 43A and 43B show CART33 treatment result in a dose dependentreduction of leukemia burdenin MOLM14 engrafted xenografts. FIG. 43A isa schematic showing an experimental set-up described in Example 6. FIG.43B shows the quantification of the tumor burden over time as measuredby bioluminescent imaging (BLI) in different groups.

FIG. 44 is a graph showing the tumor burden over time as measured bybioluminescent imaging (BLI) in the different groups in the experimentalset-up shown in FIG. 11.

FIG. 45 shows the combination of RNA-CART33 and chemotherapy result infurther reduction of leukemic burden in MOLM14 engrafted xenografts.

FIGS. 46A and 46B show the antibody binding capacity of CD33 and CD123on MOLM14 and Primary AML samples used for in vivo experiments. Assaywas performed using QUANTUM SIMPLY CELLULAR kit (Bangs Laboratories,Inc). Samples were washed in flow buffer and then stain with theindicated antibody (CD33 or CD123) conjugated to PE. The five differentmicrospheres provided in the kit were also stained with the sameantibody. The mean fluorescence intensity of the target was compared tothat of the five microspheres and the value of antibody binding capacitywas then calculated per the manufacture protocol. FIG. 46A shows theantibody binding capacity of MOLM14 for CD33 and CD123, while FIG. 46Bshows the antibody binding capacity of the primary samples used in theseexperiments for CD33 and CD123.

FIG. 47 is a graph showing the proliferation by cell count of various Tcells—CART-33 T cells (CD33-1 through CD33-9, or Upenn), CART-CD19 Tcells (CD19), or unstransduced T cells (Cell) when exposed to PL21,HL-60, or MOLP8 target cells.

FIG. 48A is a graph showing the percent of HL-60-Luc target cell lysiswhen exposed to various CART-33 T cells (CD33-1 through CD33-9, orUpenn), CART-CD19 T cells (CD19), or unstransduced T cells (Cell) atvarious effector to T cell ratios. FIG. 48B is a graph showing thepercent of PL21/Luc target cell lysis when exposed to various CART-33 Tcells (CD33-1 through CD33-9, or Upenn), CART-CD19 T cells (CD19), orunstransduced T cells (Cell) at various effector to T cell ratios. FIG.48C is a graph showing the percent of U87/Luc target cell lysis whenexposed to various CART-33 T cells (CD33-1 through CD33-9, or Upenn),CART-CD19 T cells (CD19), or unstransduced T cells (Cell) at variouseffector to T cell ratios.

FIG. 49 is a graph showing the concentration of cytokines (humaninterferon-gamma (IFN-γ), human interleukin-2 (IL-2), and human tumornecrosis factor (TNF)) produced by various CART-33 T cells (CD33-1through CD33-9, or Upenn), CART-CD19 T cells (CD19), or unstransduced Tcells (Cell) when exposed to PL21, HL60, or MOLP8 target cells.

FIG. 50A is a flow cytometry plot showing the gating strategy for MDSCs.FIG. 50B is a graph showing the percent of lineage negative, HLA-DRnegative, CD33+(LIN-HLDR-CD33+) cells in bone marrows from normal donors(ND BM) or from myelodysplastic syndrome (MDS) patients (MDS BM). FIG.50C is a graph showing the level of CD33 measured by mean fluorescenceintensity (MFI) in the MDSC population (MDSCs) compared to malignant MDSpopulation (MDS) and normal donor population (ND-BM).

FIG. 51A is a panel of flow cytometry plots showing the extent ofdegranulation (CD107a level) and cytokine production (GM-CSF, IL-2,TNF-α) from CART33. CD107a degranulation and cytokine production areshown on the y-axis, and anti-CD33 CAR on the x-axis. The negativecontrol is shown on the left (Jurkat) and the MDSC on the right. FIG.51B is a graph showing the quantification of degranulation and cytokineproduction by CART33 against various targets—Jurkat, PMA/ionomycin(PMA/IONO), MDS (non-MDSCs), and MDSCs.

FIG. 52A-52D show the various configurations on a single vector, e.g.,where the U6 regulated shRNA is upstream or downstream of the EF1 alpharegulated CAR encoding elements. In the exemplary constructs depicted inFIGS. 52A and 52B, the transcription occurs through the U6 and EF1 alphapromoters in the same direction. In the exemplary constructs depicted inFIGS. 52C and 52D, the transcription occurs through the U6 and EF1 alphapromoters in different directions. In FIG. 52E, the shRNA (andcorresponding U6 promoter) is on a first vector, and the CAR (andcorresponding EF1 alpha promoter) is on a second vector.

FIG. 53 depicts the structures of two exemplary RCAR configurations. Theantigen binding members comprise an antigen binding domain, atransmembrane domain, and a switch domain. The intracellular bindingmembers comprise a switch domain, a co-stimulatory signaling domain anda primary signaling domain. The two configurations demonstrate that thefirst and second switch domains described herein can be in differentorientations with respect to the antigen binding member and theintracellular binding member. Other RCAR configurations are furtherdescribed herein.

FIG. 54 shows that the proliferation of CAR-expressing, transduced Tcells is enhanced by low doses of RAD001 in a cell culture system. CARTswere co-cultured with Nalm-6 cells in the presence of differentconcentrations of RAD001. The number of CAR-positive CD3-positive Tcells (black) and total T cells (gray) was assessed after 4 days ofco-culture.

FIG. 55 depicts tumor growth measurements of NALM6-luc cells with dailyRAD001 dosing at 0.3, 1, 3, and 10 mg/kg (mpk) or vehicle dosing.Circles denote the vehicle; squares denote the 10 mg/kg dose of RAD001;triangles denote the 3 mg/kg dose of RAD001, inverted triangles denotethe 1 mg/kg dose of RAD001; and diamonds denote the 0.3 mg/kg dose ofRAD001.

FIGS. 56A and 56B show pharmacokinetic curves showing the amount ofRAD001 in the blood of NSG mice with NALM6 tumors. FIG. 56A shows day 0PK following the first dose of RAD001. FIG. 56B shows Day 14 PKfollowing the final RAD001 dose. Diamonds denote the 10 mg/kg dose ofRAD001; squares denote the 1 mg/kg dose of RAD001; triangles denote the3 mg/kg dose of RAD001; and x's denote the 10 mg/kg dose of RAD001.

FIGS. 57A and 57B show in vivo proliferation of humanized CD19 CARTcells with and without RAD001 dosing. Low doses of RAD001 (0.003 mg/kg)daily lead to an enhancement in CAR T cell proliferation, above thenormal level of huCAR19 proliferation. FIG. 57A shows CD4⁺ CAR T cells;FIG. 57B shows CD8⁺ CAR T cells. Circles denote PBS; squares denotehuCTL019; triangles denote huCTL019 with 3 mg/kg RAD001; invertedtriangles denote huCTL019 with 0.3 mg/kg RAD001; diamonds denotehuCTL019 with 0.03 mg/kg RAD001; and circles denote huCTL019 with 0.003mg/kg RAD001.

FIG. 58 is a graph showing HL-60-luc xenograft AML disease progression.Blue circles: mice treated with 100 ul of PBS via the tail vein; redsquares: mice treated with CD19 CAR T cells; green triangles: micetreated with CD33-1 CAR transduced T cells; inverted purple triangles:mice treated with CD33-2 CAR transduced T cells; orange diamonds: micetreated with CD33-4 CAR transduced T cells; black squares: mice treatedwith CD33-5 CAR transduced T cells; brown triangles: mice treated withCD33-6 CAR transduced T cells; dark blue circles: mice treated withCD33-7 CAR transduced T cells; and inverted dark purple triangles: micetreated with CD33-9 CAR transduced T cells.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains.

The term “a” and “an” refers to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

The term “about” when referring to a measurable value such as an amount,a temporal duration, and the like, is meant to encompass variations of±20% or in some instances ±10%, or in some instances ±5%, or in someinstances ±1%, or in some instances ±0.1% from the specified value, assuch variations are appropriate to perform the disclosed methods.

The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers toa recombinant polypeptide construct comprising at least an extracellularantigen binding domain, a transmembrane domain and a cytoplasmicsignaling domain (also referred to herein as “an intracellular signalingdomain”) comprising a functional signaling domain derived from astimulatory molecule as defined below. In some embodiments, the domainsin the CAR polypeptide construct are in the same polypeptide chain,e.g., comprise a chimeric fusion protein. In some embodiments, thedomains in the CAR polypeptide construct are not contiguous with eachother, e.g., are in different polypeptide chains, e.g., as provided inan RCAR as described herein.

In one aspect, the stimulatory molecule of the CAR is the zeta chainassociated with the T cell receptor complex. In one aspect, thecytoplasmic signaling domain comprises a primary signaling domain (e.g.,a primary signaling domain of CD3-zeta). In one aspect, the cytoplasmicsignaling domain further comprises one or more functional signalingdomains derived from at least one costimulatory molecule as definedbelow. In one aspect, the costimulatory molecule is chosen from 4-1BB(i.e., CD137), CD27, ICOS, and/or CD28. In one aspect, the CAR comprisesa chimeric fusion protein comprising an extracellular antigenrecognition domain, a transmembrane domain and an intracellularsignaling domain comprising a functional signaling domain derived from astimulatory molecule. In one aspect, the CAR comprises a chimeric fusionprotein comprising an extracellular antigen recognition domain, atransmembrane domain and an intracellular signaling domain comprising afunctional signaling domain derived from a co-stimulatory molecule and afunctional signaling domain derived from a stimulatory molecule. In oneaspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen recognition domain, a transmembrane domain and anintracellular signaling domain comprising two functional signalingdomains derived from one or more co-stimulatory molecule(s) and afunctional signaling domain derived from a stimulatory molecule. In oneaspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen recognition domain, a transmembrane domain and anintracellular signaling domain comprising at least two functionalsignaling domains derived from one or more co-stimulatory molecule(s)and a functional signaling domain derived from a stimulatory molecule.In one aspect the CAR comprises an optional leader sequence at theamino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CARfurther comprises a leader sequence at the N-terminus of theextracellular antigen recognition domain, wherein the leader sequence isoptionally cleaved from the antigen recognition domain (e.g., aa scFv)during cellular processing and localization of the CAR to the cellularmembrane.

A CAR that comprises an antigen binding domain (e.g., a scFv, a singledomain antibody, or TCR (e.g., a TCR alpha binding domain or TCR betabinding domain)) that targets a specific tumor marker X, wherein X canbe a tumor marker as described herein, is also referred to as XCAR. Forexample, a CAR that comprises an antigen binding domain that targetsCD33 is referred to as CD33CAR. The CAR can be expressed in any cell,e.g., an immune effector cell as described herein (e.g., a T cell or anNK cell).

The term “signaling domain” refers to the functional portion of aprotein which acts by transmitting information within the cell toregulate cellular activity via defined signaling pathways by generatingsecond messengers or functioning as effectors by responding to suchmessengers.

As used herein, the term “CD33” refers to the Cluster of Differentiation33 protein, which is an antigenic determinant detectable on leukemiacells as well on normal precursor cells of the myeloid lineage. Thehuman and murine amino acid and nucleic acid sequences can be found in apublic database, such as GenBank, UniProt and Swiss-Prot. For example,the amino acid sequence of human CD33 can be found as UniProt/Swiss-ProtAccession No. P20138 and the nucleotide sequence encoding of the humanCD33 can be found at Accession No. NM_001772.3. In one aspect theantigen-binding portion of the CAR recognizes and binds an epitopewithin the extracellular domain of the CD33 protein or fragmentsthereof. In one aspect, the CD33 protein is expressed on a cancer cell.As used herein, “CD33” includes proteins comprising mutations, e.g.,point mutations, fragments, insertions, deletions and splice variants offull length wild-type CD33.

The term “antibody,” as used herein, refers to a protein, or polypeptidesequence derived from an immunoglobulin molecule which specificallybinds with an antigen. Antibodies can be polyclonal or monoclonal,multiple or single chain, or intact immunoglobulins, and may be derivedfrom natural sources or from recombinant sources. Antibodies can betetramers of immunoglobulin molecules.

The term “antibody fragment” refers to at least one portion of an intactantibody, or recombinant variants thereof, and refers to the antigenbinding domain, e.g., an antigenic determining variable region of anintact antibody, that is sufficient to confer recognition and specificbinding of the antibody fragment to a target, such as an antigen.Examples of antibody fragments include, but are not limited to, Fab,Fab, F(ab)₂, and Fv fragments, scFv antibody fragments, linearantibodies, single domain antibodies such as sdAb (either VL or VH),camelid VHH domains, and multi-specific molecules formed from antibodyfragments such as a bivalent fragment comprising two or more, e.g., two,Fab fragments linked by a disulfide brudge at the hinge region, or twoor more, e.g., two isolated CDR or other epitope binding fragments of anantibody linked. An antibody fragment can also be incorporated intosingle domain antibodies, maxibodies, minibodies, nanobodies,intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv(see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136,2005). Antibody fragments can also be grafted into scaffolds based onpolypeptides such as a fibronectin type III (Fn3)(see U.S. Pat. No.6,703,199, which describes fibronectin polypeptide minibodies).

The term “scFv” refers to a fusion protein comprising at least oneantibody fragment comprising a variable region of a light chain and atleast one antibody fragment comprising a variable region of a heavychain, wherein the light and heavy chain variable regions arecontiguously linked via a short flexible polypeptide linker, and capableof being expressed as a single chain polypeptide, and wherein the scFvretains the specificity of the intact antibody from which it is derived.Unless specified, as used herein an scFv may have the VL and VH variableregions in either order, e.g., with respect to the N-terminal andC-terminal ends of the polypeptide, the scFv may comprise VL-linker-VHor may comprise VH-linker-VL.

The terms “complementarity determining region” or “CDR,” as used herein,refer to the sequences of amino acids within antibody variable regionswhich confer antigen specificity and binding affinity. For example, ingeneral, there are three CDRs in each heavy chain variable region (e.g.,HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variableregion (LCDR1, LCDR2, and LCDR3). The precise amino acid sequenceboundaries of a given CDR can be determined using any of a number ofwell-known schemes, including those described by Kabat et al. (1991),“Sequences of Proteins of Immunological Interest,” 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (“Kabat” numberingscheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numberingscheme), or a combination thereof. Under the Kabat numbering scheme, insome embodiments, the CDR amino acid residues in the heavy chainvariable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and95-102 (HCDR3); and the CDR amino acid residues in the light chainvariable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments,the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2),and 95-102 (HCDR3); and the CDR amino acid residues in the VL arenumbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combinedKabat and Chothia numbering scheme, in some embodiments, the CDRscorrespond to the amino acid residues that are part of a Kabat CDR, aChothia CDR, or both. For instance, in some embodiments, the CDRscorrespond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and95-102 (HCDR3) in a VH, e.g., a mammalian VH, e.g., a human VH; andamino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in aVL, e.g., a mammalian VL, e.g., a human VL.

The portion of the CAR composition of the invention comprising anantibody or antibody fragment thereof may exist in a variety of forms,for example, where the antigen binding domain is expressed as part of apolypeptide chain including, for example, a single domain antibodyfragment (sdAb), a single chain antibody (scFv), or e.g., a humanized orhuman antibody (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426). In one aspect, the antigen binding domain ofa CAR composition of the invention comprises an antibody fragment. In afurther aspect, the CAR comprises an antibody fragment that comprises ascFv.

As used herein, the term “binding domain” or “antibody molecule” refersto a protein, e.g., an immunoglobulin chain or fragment thereof,comprising at least one immunoglobulin variable domain sequence. Theterm “binding domain” or “antibody molecule” encompasses antibodies andantibody fragments. In an embodiment, an antibody molecule is amultispecific antibody molecule, e.g., it comprises a plurality ofimmunoglobulin variable domain sequences, wherein a first immunoglobulinvariable domain sequence of the plurality has binding specificity for afirst epitope and a second immunoglobulin variable domain sequence ofthe plurality has binding specificity for a second epitope. In anembodiment, a multispecific antibody molecule is a bispecific antibodymolecule. A bispecific antibody has specificity for no more than twoantigens. A bispecific antibody molecule is characterized by a firstimmunoglobulin variable domain sequence which has binding specificityfor a first epitope and a second immunoglobulin variable domain sequencethat has binding specificity for a second epitope.

The term “antibody heavy chain,” refers to the larger of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations, and which normally determines the class towhich the antibody belongs.

The term “antibody light chain,” refers to the smaller of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations. Kappa (κ) and lambda (λ) light chains refer tothe two major antibody light chain isotypes.

The term “recombinant antibody” refers to an antibody which is generatedusing recombinant DNA technology, such as, for example, an antibodyexpressed by a bacteriophage or yeast expression system. The term shouldalso be construed to mean an antibody which has been generated by thesynthesis of a DNA molecule encoding the antibody and which DNA moleculeexpresses an antibody protein, or an amino acid sequence specifying theantibody, wherein the DNA or amino acid sequence has been obtained usingrecombinant DNA or amino acid sequence technology which is available andwell known in the art.

The term “antigen” or “Ag” refers to a molecule that provokes an immuneresponse. This immune response may involve either antibody production,or the activation of specific immunologically-competent cells, or both.The skilled artisan will understand that any macromolecule, includingvirtually all proteins or peptides, can serve as an antigen.Furthermore, antigens can be derived from recombinant or genomic DNA. Askilled artisan will understand that any DNA, which comprises anucleotide sequences or a partial nucleotide sequence encoding a proteinthat elicits an immune response therefore encodes an “antigen” as thatterm is used herein. Furthermore, one skilled in the art will understandthat an antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to encode polypeptides that elicit the desiredimmune response. Moreover, a skilled artisan will understand that anantigen need not be encoded by a “gene” at all. It is readily apparentthat an antigen can be generated synthesized or can be derived from abiological sample, or might be macromolecule besides a polypeptide. Sucha biological sample can include, but is not limited to a tissue sample,a tumor sample, a cell or a fluid with other biological components.

The term “anti-tumor effect” refers to a biological effect which can bemanifested by various means, including but not limited to, e.g., adecrease in tumor volume, a decrease in the number of tumor cells, adecrease in the number of metastases, an increase in life expectancy,decrease in tumor cell proliferation, decrease in tumor cell survival,or amelioration of various physiological symptoms associated with thecancerous condition. An “anti-tumor effect” can also be manifested bythe ability of the peptides, polynucleotides, cells and antibodies ofthe invention in prevention of the occurrence of tumor in the firstplace.

The term “anti-cancer effect” refers to a biological effect which can bemanifested by various means, including but not limited to, e.g., adecrease in tumor volume, a decrease in the number of cancer cells, adecrease in the number of metastases, an increase in life expectancy,decrease in cancer cell proliferation, decrease in cancer cell survival,or amelioration of various physiological symptoms associated with thecancerous condition. An “anti-cancer effect” can also be manifested bythe ability of the peptides, polynucleotides, cells and antibodies inprevention of the occurrence of cancer in the first place. The term“anti-tumor effect” refers to a biological effect which can bemanifested by various means, including but not limited to, e.g., adecrease in tumor volume, a decrease in the number of tumor cells, adecrease in tumor cell proliferation, or a decrease in tumor cellsurvival.

The term “autologous” refers to any material derived from the sameindividual to whom it is later to be re-introduced into the individual.

The term “allogeneic” refers to any material derived from a differentanimal of the same species as the individual to whom the material isintroduced. Two or more individuals are said to be allogeneic to oneanother when the genes at one or more loci are not identical. In someaspects, allogeneic material from individuals of the same species may besufficiently unlike genetically to interact antigenically.

The term “xenogeneic” refers to a graft derived from an animal of adifferent species.

The term “apheresis” as used herein refers to the art-recognizedextracorporeal process by which the blood of a donor or patient isremoved from the donor or patient and passed through an apparatus thatseparates out selected particular constituent(s) and returns theremainder to the circulation of the donor or patient, e.g., byretransfusion. Thus, in the context of “an apheresis sample” refers to asample obtained using apheresis.

The term “combination” refers to either a fixed combination in onedosage unit form, or a combined administration where a compound of thepresent invention and a combination partner (e.g. another drug asexplained below, also referred to as “therapeutic agent” or “co-agent”)may be administered independently at the same time or separately withintime intervals, especially where these time intervals allow that thecombination partners show a cooperative, e.g. synergistic effect. Thesingle components may be packaged in a kit or separately. One or both ofthe components (e.g., powders or liquids) may be reconstituted ordiluted to a desired dose prior to administration. The terms“co-administration” or “combined administration” or the like as utilizedherein are meant to encompass administration of the selected combinationpartner to a single subject in need thereof (e.g. a patient), and areintended to include treatment regimens in which the agents are notnecessarily administered by the same route of administration or at thesame time. The term “pharmaceutical combination” as used herein means aproduct that results from the mixing or combining of more than oneactive ingredient and includes both fixed and non-fixed combinations ofthe active ingredients. The term “fixed combination” means that theactive ingredients, e.g. a compound of the present invention and acombination partner, are both administered to a patient simultaneouslyin the form of a single entity or dosage. The term “non-fixedcombination” means that the active ingredients, e.g. a compound of thepresent invention and a combination partner, are both administered to apatient as separate entities either simultaneously, concurrently orsequentially with no specific time limits, wherein such administrationprovides therapeutically effective levels of the two compounds in thebody of the patient. The latter also applies to cocktail therapy, e.g.the administration of three or more active ingredients.

The term “cancer” refers to a disease characterized by the rapid anduncontrolled growth of aberrant cells. Cancer cells can spread locallyor through the bloodstream and lymphatic system to other parts of thebody. Examples of various cancers are described herein and include butare not limited to, breast cancer, prostate cancer, ovarian cancer,cervical cancer, skin cancer, pancreatic cancer, colorectal cancer,renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lungcancer and the like. The terms “tumor” and “cancer” are usedinterchangeably herein, e.g., both terms encompass solid and liquid,e.g., diffuse or circulating, tumors. As used herein, the term “cancer”or “tumor” includes premalignant, as well as malignant cancers andtumors.

“Derived from” as that term is used herein, indicates a relationshipbetween a first and a second molecule. It generally refers to structuralsimilarity between the first molecule and a second molecule and does notconnotate or include a process or source limitation on a first moleculethat is derived from a second molecule. For example, in the case of anintracellular signaling domain that is derived from a CD3zeta molecule,the intracellular signaling domain retains sufficient CD3zeta structuresuch that is has the required function, namely, the ability to generatea signal under the appropriate conditions. It does not connotate orinclude a limitation to a particular process of producing theintracellular signaling domain, e.g., it does not mean that, to providethe intracellular signaling domain, one must start with a CD3zetasequence and delete unwanted sequence, or impose mutations, to arrive atthe intracellular signaling domain.

The phrase “disease associated with expression of CD33” as used hereinincludes but is not limited to, a disease associated with a cell whichexpresses CD33 (e.g., wild-type or mutant CD33) or condition associatedwith a cell which expresses CD33 (e.g., wild-type or mutant CD33)including, e.g., a proliferative disease such as a cancer or malignancyor a precancerous condition such as a myelodysplasia, a myelodysplasticsyndrome or a preleukemia; or a noncancer related indication associatedwith a cell which expresses CD33 (e.g., wild-type or mutant CD33). Forthe avoidance of doubt, a disease associated with expression of CD33 mayinclude a condition associated with a cell which do not presentlyexpress CD33, e.g., because CD33 expression has been downregulated,e.g., due to treatment with a molecule targeting CD33, e.g., a CD33inhibitor described herein, but which at one time expressed CD33. In oneaspect, a cancer associated with expression of CD33 (e.g., wild-type ormutant CD33) is a hematological cancer. In one aspect, a hematologicalcancer includes but is not limited to acute myeloid leukemia (AML),myelodysplasia and myelodysplastic syndrome, myelofibrosis andmyeloproliferative neoplasms, acute lymphoid leukemia (ALL), hairy cellleukemia, Prolymphocytic leukemia, chronic myeloid leukemia (CIVIL),Blastic plasmacytoid dendritic cell neoplasm, and the like. Furtherdisease associated with expression of CD33 (e.g., wild-type or mutantCD33) expression include, but are not limited to, e.g., atypical and/ornon-classical cancers, malignancies, precancerous conditions orproliferative diseases associated with expression of CD33 (e.g.,wild-type or mutant CD33). Non-cancer related indications associatedwith expression of CD33 (e.g., wild-type or mutant CD33) may also beincluded. In embodiments, a non-cancer related indication associatedwith expression of CD33 includes but is not limited to, e.g., autoimmunedisease, (e.g., lupus), inflammatory disorders (allergy and asthma) andtransplantation. In some embodiments, the tumor antigen-expressing cellexpresses, or at any time expressed, mRNA encoding the tumor antigen. Inan embodiment, the tumor antigen-expressing cell produces the tumorantigen protein (e.g., wild-type or mutant), and the tumor antigenprotein may be present at normal levels or reduced levels. In anembodiment, the tumor antigen-expressing cell produced detectable levelsof a tumor antigen protein at one point, and subsequently producedsubstantially no detectable tumor antigen protein.

The term “conservative sequence modifications” refers to amino acidmodifications that do not significantly affect or alter the bindingcharacteristics of the antibody or antibody fragment containing theamino acid sequence. Such conservative modifications include amino acidsubstitutions, additions and deletions. Modifications can be introducedinto an antibody or antibody fragment of the invention by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. Conservative substitutions are ones in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within a CAR of the invention can bereplaced with other amino acid residues from the same side chain familyand the altered CAR can be tested using the functional assays describedherein.

The term “stimulation,” refers to a primary response induced by bindingof a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognateligand thereby mediating a signal transduction event, such as, but notlimited to, signal transduction via the TCR/CD3 complex. Stimulation canmediate altered expression of certain molecules, such as downregulationof TGF-β, and/or reorganization of cytoskeletal structures, and thelike.

The term “stimulatory molecule,” refers to a molecule expressed by a Tcell that provides the primary cytoplasmic signaling sequence(s) thatregulate primary activation of the TCR complex in a stimulatory way forat least some aspect of the T cell signaling pathway. In one aspect, theprimary signal is initiated by, for instance, binding of a TCR/CD3complex with an MHC molecule loaded with peptide, and which leads tomediation of a T cell response, including, but not limited to,proliferation, activation, differentiation, and the like. A primarycytoplasmic signaling sequence (also referred to as a “primary signalingdomain”) that acts in a stimulatory manner may contain a signaling motifwhich is known as immunoreceptor tyrosine-based activation motif orITAM. Examples of an ITAM containing primary cytoplasmic signalingsequence that is of particular use in the invention includes, but is notlimited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma,CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as“ICOS”), FcεRI and CD66d, DAP10 and DAP12. In a specific CAR of theinvention, the intracellular signaling domain in any one or more CARs ofthe invention comprises an intracellular signaling sequence, e.g., aprimary signaling sequence of CD3-zeta. In a specific CAR of theinvention, the primary signaling sequence of CD3-zeta is the sequenceprovided as SEQ ID NO:9, or the equivalent residues from a non-humanspecies, e.g., mouse, rodent, monkey, ape and the like. In a specificCAR of the invention, the primary signaling sequence of CD3-zeta is thesequence as provided in SEQ ID NO:10, or the equivalent residues from anon-human species, e.g., mouse, rodent, monkey, ape and the like.

The term “antigen presenting cell” or “APC” refers to an immune systemcell such as an accessory cell (e.g., a B-cell, a dendritic cell, andthe like) that displays a foreign antigen complexed with majorhistocompatibility complexes (MHC on its surface. T-cells may recognizethese complexes using their T-cell receptors (TCRs). APCs processantigens and present them to T-cells.

An “intracellular signaling domain,” as the term is used herein, refersto an intracellular portion of a molecule. In embodiments, theintracellular signal domain transduces the effector function signal anddirects the cell to perform a specialized function. While the entireintracellular signaling domain can be employed, in many cases it is notnecessary to use the entire chain. To the extent that a truncatedportion of the intracellular signaling domain is used, such truncatedportion may be used in place of the intact chain as long as ittransduces the effector function signal. The term intracellularsignaling domain is thus meant to include any truncated portion of theintracellular signaling domain sufficient to transduce the effectorfunction signal. The intracellular signaling domain can generate asignal that promotes an immune effector function of the CAR containingcell, e.g., a CART cell or CAR-expressing NK cell. Examples of immuneeffector function, e.g., in a CART cell or CAR-expressing NK cell,include cytolytic activity and helper activity, including the secretionof cytokines.

In an embodiment, the intracellular signaling domain can comprise aprimary intracellular signaling domain. Exemplary primary intracellularsignaling domains include those derived from the molecules responsiblefor primary stimulation, or antigen dependent simulation. In anembodiment, the intracellular signaling domain can comprise acostimulatory intracellular domain. Exemplary costimulatoryintracellular signaling domains include those derived from moleculesresponsible for costimulatory signals, or antigen independentstimulation. For example, in the case of a CAR-expressing immuneeffector cell, e.g., CART cell or CAR-expressing NK cell, a primaryintracellular signaling domain can comprise a cytoplasmic sequence of aT cell receptor, and a costimulatory intracellular signaling domain cancomprise cytoplasmic sequence from co-receptor or costimulatorymolecule.

A primary intracellular signaling domain can comprise a signaling motifwhich is known as an immunoreceptor tyrosine-based activation motif orITAM. Examples of ITAM containing primary cytoplasmic signalingsequences include, but are not limited to, those derived from CD3 zeta,FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, CD278 (also known as “ICOS”), FcεRI and CD66d, DAP10 andDAP12.

The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta”is defined as the protein provided as GenBan Acc. No. BAG36664.1, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like, and a “zeta stimulatory domain” oralternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatorydomain” is defined as the amino acid residues from the cytoplasmicdomain of the zeta chain that are sufficient to functionally transmit aninitial signal necessary for T cell activation. In one aspect thecytoplasmic domain of zeta comprises residues 52 through 164 of GenBankAcc. No. BAG36664.1 or the equivalent residues from a non-human species,e.g., mouse, rodent, monkey, ape and the like, that are functionalorthologs thereof. In one aspect, the “zeta stimulatory domain” or a“CD3-zeta stimulatory domain” is the sequence provided as SEQ ID NO:9.In one aspect, the “zeta stimulatory domain” or a “CD3-zeta stimulatorydomain” is the sequence provided as SEQ ID NO:10.

The term “costimulatory molecule” refers to the cognate binding partneron a T cell that specifically binds with a costimulatory ligand, therebymediating a costimulatory response by the T cell, such as, but notlimited to, proliferation. Costimulatory molecules are cell surfacemolecules other than antigen receptors or their ligands that arerequired for an efficient immune response. Costimulatory moleculesinclude, but are not limited to an MHC class I molecule, TNF receptorproteins, Immunoglobulin-like proteins, cytokine receptors, integrins,signaling lymphocytic activation molecules (SLAM proteins), activatingNK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27,CD28, CD30, CD40, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3,CD5, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

A costimulatory intracellular signaling domain refers to theintracellular portion of a costimulatory molecule.

The intracellular signaling domain can comprise the entire intracellularportion, or the entire native intracellular signaling domain, of themolecule from which it is derived, or a functional fragment thereof.

The term “4-1BB” refers to a member of the TNFR superfamily with anamino acid sequence provided as GenBank Acc. No. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like; and a “4-1BB costimulatory domain” is definedas amino acid residues 214-255 of GenBank accno. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like. In one aspect, the “4-1BB costimulatorydomain” is the sequence provided as SEQ ID NO:7 or the equivalentresidues from a non-human species, e.g., mouse, rodent, monkey, ape andthe like.

“Immune effector cell,” as that term is used herein, refers to a cellthat is involved in an immune response, e.g., in the promotion of animmune effector response. Examples of immune effector cells include Tcells, e.g., alpha/beta T cells and gamma/delta T cells, B cells,natural killer (NK) cells, natural killer T (NKT) cells, mast cells, andmyeloic-derived phagocytes.

“Immune effector function or immune effector response,” as that term isused herein, refers to function or response, e.g., of an immune effectorcell, that enhances or promotes an immune attack of a target cell. E.g.,an immune effector function or response refers a property of a T or NKcell that promotes killing or the inhibition of growth or proliferation,of a target cell. In the case of a T cell, primary stimulation andco-stimulation are examples of immune effector function or response.

The term “effector function” refers to a specialized function of a cell.Effector function of a T cell, for example, may be cytolytic activity orhelper activity including the secretion of cytokines.

The term “encoding” refers to the inherent property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as templates for synthesis of other polymers andmacromolecules in biological processes having either a defined sequenceof nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence ofamino acids and the biological properties resulting therefrom. Thus, agene, cDNA, or RNA, encodes a protein if transcription and translationof mRNA corresponding to that gene produces the protein in a cell orother biological system. Both the coding strand, the nucleotide sequenceof which is identical to the mRNA sequence and is usually provided insequence listings, and the non-coding strand, used as the template fortranscription of a gene or cDNA, can be referred to as encoding theprotein or other product of that gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or a RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “effective amount” or “therapeutically effective amount” areused interchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result. The term “endogenous” refers toany material from or produced inside an organism, cell, tissue orsystem.

The term “exogenous” refers to any material introduced from or producedoutside an organism, cell, tissue or system.

The term “expression” refers to the transcription and/or translation ofa particular nucleotide sequence driven by a promoter.

The term “transfer vector” refers to a composition of matter whichcomprises an isolated nucleic acid and which can be used to deliver theisolated nucleic acid to the interior of a cell. Numerous vectors areknown in the art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “transfer vector” includes anautonomously replicating plasmid or a virus. The term should also beconstrued to further include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example, apolylysine compound, liposome, and the like. Examples of viral transfervectors include, but are not limited to, adenoviral vectors,adeno-associated virus vectors, retroviral vectors, lentiviral vectors,and the like.

The term “expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, including cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “lentivirus” refers to a genus of the Retroviridae family.Lentiviruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. HIV, SIV, and FIV are allexamples of lentiviruses.

The term “lentiviral vector” refers to a vector derived from at least aportion of a lentivirus genome, including especially a self-inactivatinglentiviral vector as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentivirus vectors that may be usedin the clinic, include but are not limited to, e.g., the LENTIVECTOR®gene delivery technology from Oxford BioMedica, the LENTIMAX™ vectorsystem from Lentigen and the like. Nonclinical types of lentiviralvectors are also available and would be known to one skilled in the art.

The term “homologous” or “identity” refers to the subunit sequenceidentity between two polymeric molecules, e.g., between two nucleic acidmolecules, such as, two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous or identical at that position. The homology between twosequences is a direct function of the number of matching or homologouspositions; e.g., if half (e.g., five positions in a polymer ten subunitsin length) of the positions in two sequences are homologous, the twosequences are 50% homologous; if 90% of the positions (e.g., 9 of 10),are matched or homologous, the two sequences are 90% homologous.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab, F(ab)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies and antibody fragments thereofare human immunoglobulins (recipient antibody or antibody fragment) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, a humanizedantibody/antibody fragment can comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. These modifications can further refine and optimize antibodyor antibody fragment performance. In general, the humanized antibody orantibody fragment thereof will comprise substantially all of at leastone, and typically two, variable domains, in which all or substantiallyall of the CDR regions correspond to those of a non-human immunoglobulinand all or a significant portion of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody or antibody fragment canalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin. For further details, seeJones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

“Fully human” refers to an immunoglobulin, such as an antibody orantibody fragment, where the whole molecule is of human origin orconsists of an amino acid sequence identical to a human form of theantibody or immunoglobulin.

The term “isolated” means altered or removed from the natural state. Forexample, a nucleic acid or a peptide naturally present in a livinganimal is not “isolated,” but the same nucleic acid or peptide partiallyor completely separated from the coexisting materials of its naturalstate is “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The term “operably linked” or “transcriptional control” refers tofunctional linkage between a regulatory sequence and a heterologousnucleic acid sequence resulting in expression of the latter. Forexample, a first nucleic acid sequence is operably linked with a secondnucleic acid sequence when the first nucleic acid sequence is placed ina functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Operably linked DNA sequences can be contiguous with each other and,e.g., where necessary to join two protein coding regions, are in thesame reading frame.

The term “parenteral” administration of an immunogenic compositionincludes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular(i.m.), or intrasternal injection, intratumoral, or infusion techniques.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)).

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. A polypeptide includes a natural peptide, arecombinant peptide, or a combination thereof.

The term “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required toinitiate the specific transcription of a polynucleotide sequence.

The term “promoter/regulatory sequence” refers to a nucleic acidsequence which is required for expression of a gene product operablylinked to the promoter/regulatory sequence. In some instances, thissequence may be the core promoter sequence and in other instances, thissequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

The term “constitutive” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cell undermost or all physiological conditions of the cell.

The term “inducible” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

The term “tissue-specific” promoter refers to a nucleotide sequencewhich, when operably linked with a polynucleotide encodes or specifiedby a gene, causes the gene product to be produced in a cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

The terms “cancer associated antigen” or “tumor antigen” interchangeablyrefers to a molecule (typically a protein, carbohydrate or lipid) thatis expressed on the surface of a cancer cell, either entirely or as afragment (e.g., MHC/peptide), and which is useful for the preferentialtargeting of a pharmacological agent to the cancer cell. In someembodiments, a tumor antigen is a marker expressed by both normal cellsand cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In someembodiments, a tumor antigen is a cell surface molecule that isoverexpressed in a cancer cell in comparison to a normal cell, forinstance, 1-fold over expression, 2-fold overexpression, 3-foldoverexpression or more in comparison to a normal cell. In someembodiments, a tumor antigen is a cell surface molecule that isinappropriately synthesized in the cancer cell, for instance, a moleculethat contains deletions, additions or mutations in comparison to themolecule expressed on a normal cell. In some embodiments, a tumorantigen will be expressed exclusively on the cell surface of a cancercell, entirely or as a fragment (e.g., MHC/peptide), and not synthesizedor expressed on the surface of a normal cell. In some embodiments, theCARs of the present invention includes CARs comprising an antigenbinding domain (e.g., antibody or antibody fragment) that binds to a MHCpresented peptide. Normally, peptides derived from endogenous proteinsfill the pockets of Major histocompatibility complex (MHC) class Imolecules, and are recognized by T cell receptors (TCRs) on CD8+ Tlymphocytes. The MEW class I complexes are constitutively expressed byall nucleated cells. In cancer, virus-specific and/or tumor-specificpeptide/MHC complexes represent a unique class of cell surface targetsfor immunotherapy. TCR-like antibodies targeting peptides derived fromviral or tumor antigens in the context of human leukocyte antigen(HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., JVirol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165;Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci TranslMed 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 201219(2):84-100). For example, TCR-like antibody can be identified fromscreening a library, such as a human scFv phage displayed library.

The term “tumor-supporting antigen” or “cancer-supporting antigen”interchangeably refer to a molecule (typically a protein, carbohydrateor lipid) that is expressed on the surface of a cell that is, itself,not cancerous, but supports the cancer cells, e.g., by promoting theirgrowth or survival e.g., resistance to immune cells. Exemplary cells ofthis type include stromal cells and myeloid-derived suppressor cells(MDSCs). The tumor-supporting antigen itself need not play a role insupporting the tumor cells so long as the antigen is present on a cellthat supports cancer cells.

The term “flexible polypeptide linker” or “linker” as used in thecontext of a scFv refers to a peptide linker that consists of aminoacids such as glycine and/or serine residues used alone or incombination, to link variable heavy and variable light chain regionstogether. In one embodiment, the flexible polypeptide linker is aGly/Ser linker and comprises the amino acid sequence(Gly-Gly-Gly-Ser)_(n)(SEQ ID NO: 38), where n is a positive integerequal to or greater than 1. For example, n=1, n=2, n=3. n=4, n=5 andn=6, n=7, n=8, n=9 and n=10 In one embodiment, the flexible polypeptidelinkers include, but are not limited to, (Gly₄ Ser)₄ (SEQ ID NO:27) or(Gly₄ Ser)₃ (SEQ ID NO:28). In another embodiment, the linkers includemultiple repeats of (Gly₂Ser), (GlySer) or (Gly₃Ser) (SEQ ID NO:29).Also included within the scope of the invention are linkers described inWO2012/138475, incorporated herein by reference.

As used herein, a 5 cap (also termed an RNA cap, an RNA7-methylguanosine cap or an RNA m⁷G cap) is a modified guaninenucleotide that has been added to the “front” or 5′ end of a eukaryoticmessenger RNA shortly after the start of transcription. The 5 capconsists of a terminal group which is linked to the first transcribednucleotide. Its presence is critical for recognition by the ribosome andprotection from RNases. Cap addition is coupled to transcription, andoccurs co-transcriptionally, such that each influences the other.Shortly after the start of transcription, the 5 end of the mRNA beingsynthesized is bound by a cap-synthesizing complex associated with RNApolymerase. This enzymatic complex catalyzes the chemical reactions thatare required for mRNA capping. Synthesis proceeds as a multi-stepbiochemical reaction. The capping moiety can be modified to modulatefunctionality of mRNA such as its stability or efficiency oftranslation.

As used herein, “in vitro transcribed RNA” refers to RNA, preferablymRNA, that has been synthesized in vitro. Generally, the in vitrotranscribed RNA is generated from an in vitro transcription vector. Thein vitro transcription vector comprises a template that is used togenerate the in vitro transcribed RNA.

As used herein, a “poly(A)” is a series of adenosines attached bypolyadenylation to the mRNA. In the preferred embodiment of a constructfor transient expression, the polyA is between 50 and 5000 (SEQ ID NO:30), preferably greater than 64, more preferably greater than 100, mostpreferably greater than 300 or 400. poly(A) sequences can be modifiedchemically or enzymatically to modulate mRNA functionality such aslocalization, stability or efficiency of translation.

As used herein, “polyadenylation” refers to the covalent linkage of apolyadenylyl moiety, or its modified variant, to a messenger RNAmolecule. In eukaryotic organisms, most messenger RNA (mRNA) moleculesare polyadenylated at the 3 □end. The 3 □poly(A) tail is a long sequenceof adenine nucleotides (often several hundred) added to the pre-mRNAthrough the action of an enzyme, polyadenylate polymerase. In highereukaryotes, the poly(A) tail is added onto transcripts that contain aspecific sequence, the polyadenylation signal. The poly(A) tail and theprotein bound to it aid in protecting mRNA from degradation byexonucleases. Polyadenylation is also important for transcriptiontermination, export of the mRNA from the nucleus, and translation.Polyadenylation occurs in the nucleus immediately after transcription ofDNA into RNA, but additionally can also occur later in the cytoplasm.After transcription has been terminated, the mRNA chain is cleavedthrough the action of an endonuclease complex associated with RNApolymerase. The cleavage site is usually characterized by the presenceof the base sequence AAUAAA near the cleavage site. After the mRNA hasbeen cleaved, adenosine residues are added to the free 3 end at thecleavage site.

As used herein, “transient” refers to expression of a non-integratedtransgene for a period of hours, days or weeks, wherein the period oftime of expression is less than the period of time for expression of thegene if integrated into the genome or contained within a stable plasmidreplicon in the host cell.

As used herein, the terms “treat”, “treatment” and “treating” refer tothe reduction or amelioration of the progression, severity and/orduration of a proliferative disorder, or the amelioration of one or moresymptoms (preferably, one or more discernible symptoms) of aproliferative disorder resulting from the administration of one or moretherapies (e.g., one or more therapeutic agents such as a CAR of theinvention). In specific embodiments, the terms “treat”, “treatment” and“treating” refer to the amelioration of at least one measurable physicalparameter of a proliferative disorder, such as growth of a tumor, notnecessarily discernible by the patient. In other embodiments the terms“treat”, “treatment” and “treating”-refer to the inhibition of theprogression of a proliferative disorder, either physically by, e.g.,stabilization of a discernible symptom, physiologically by, e.g.,stabilization of a physical parameter, or both. In other embodiments theterms “treat”, “treatment” and “treating” refer to the reduction orstabilization of tumor size or cancerous cell count.

The term “signal transduction pathway” refers to the biochemicalrelationship between a variety of signal transduction molecules thatplay a role in the transmission of a signal from one portion of a cellto another portion of a cell. The phrase “cell surface receptor”includes molecules and complexes of molecules capable of receiving asignal and transmitting signal across the membrane of a cell.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals, human).

The term, a “substantially purified” cell refers to a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some aspects, thecells are cultured in vitro. In other aspects, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment. A therapeuticeffect is obtained by reduction, suppression, remission, or eradicationof a disease state.

The term “prophylaxis” as used herein means the prevention of orprotective treatment for a disease or disease state.

In the context of the present invention, “tumor antigen” or“hyperproliferative disorder antigen” or “antigen associated with ahyperproliferative disorder” refers to antigens that are common tospecific hyperproliferative disorders. In certain aspects, thehyperproliferative disorder antigens of the present invention arederived from, cancers including but not limited to primary or metastaticmelanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer,non-Hodgkin lymphoma, Hodgkin lymphoma, leukemias, uterine cancer,cervical cancer, bladder cancer, kidney cancer and adenocarcinomas suchas breast cancer, prostate cancer, ovarian cancer, pancreatic cancer,and the like.

The term “transfected” or “transformed” or “transduced” refers to aprocess by which exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny.

The term “specifically binds,” refers to an antibody, or a ligand, whichrecognizes and binds with a cognate binding partner (e.g., a stimulatoryand/or costimulatory molecule present on a T cell) protein present in asample, but which antibody or ligand does not substantially recognize orbind other molecules in the sample.

“Regulatable chimeric antigen receptor (RCAR),” as used herein, refersto a set of polypeptides, typically two in the simplest embodiments,which when in an immune effector cell, provides the cell withspecificity for a target cell, typically a cancer cell, and withintracellular signal generation. In some embodiments, an RCAR comprisesat least an extracellular antigen binding domain, a transmembrane domainand a cytoplasmic signaling domain (also referred to herein as “anintracellular signaling domain”) comprising a functional signalingdomain derived from a stimulatory molecule and/or costimulatory moleculeas defined herein in the context of a CAR molecule. In some embodiments,the set of polypeptides in the RCAR are not contiguous with each other,e.g., are in different polypeptide chains. In some embodiments, the RCARincludes a dimerization switch that, upon the presence of a dimerizationmolecule, can couple the polypeptides to one another, e.g., can couplean antigen binding domain to an intracellular signaling domain. In someembodiments, the RCAR is expressed in a cell (e.g., an immune effectorcell) as described herein, e.g., an RCAR-expressing cell (also referredto herein as “RCARX cell”). In an embodiment the RCARX cell is a T cell,and is referred to as a RCART cell. In an embodiment the RCARX cell isan NK cell, and is referred to as a RCARN cell. The RCAR can provide theRCAR-expressing cell with specificity for a target cell, typically acancer cell, and with regulatable intracellular signal generation orproliferation, which can optimize an immune effector property of theRCAR-expressing cell. In embodiments, an RCAR cell relies at least inpart, on an antigen binding domain to provide specificity to a targetcell that comprises the antigen bound by the antigen binding domain.

“Membrane anchor” or “membrane tethering domain”, as that term is usedherein, refers to a polypeptide or moiety, e.g., a myristoyl group,sufficient to anchor an extracellular or intracellular domain to theplasma membrane.

“Switch domain,” as that term is used herein, e.g., when referring to anRCAR, refers to an entity, typically a polypeptide-based entity, that,in the presence of a dimerization molecule, associates with anotherswitch domain. The association results in a functional coupling of afirst entity linked to, e.g., fused to, a first switch domain, and asecond entity linked to, e.g., fused to, a second switch domain. A firstand second switch domain are collectively referred to as a dimerizationswitch. In embodiments, the first and second switch domains are the sameas one another, e.g., they are polypeptides having the same primaryamino acid sequence, and are referred to collectively as ahomodimerization switch. In embodiments, the first and second switchdomains are different from one another, e.g., they are polypeptideshaving different primary amino acid sequences, and are referred tocollectively as a heterodimerization switch. In embodiments, the switchis intracellular. In embodiments, the switch is extracellular. Inembodiments, the switch domain is a polypeptide-based entity, e.g., FKBPor FRB-based, and the dimerization molecule is small molecule, e.g., arapalogue. In embodiments, the switch domain is a polypeptide-basedentity, e.g., an scFv that binds a myc peptide, and the dimerizationmolecule is a polypeptide, a fragment thereof, or a multimer of apolypeptide, e.g., a myc ligand or multimers of a myc ligand that bindto one or more myc scFvs. In embodiments, the switch domain is apolypeptide-based entity, e.g., myc receptor, and the dimerizationmolecule is an antibody or fragments thereof, e.g., myc antibody.

“Dimerization molecule,” as that term is used herein, e.g., whenreferring to an RCAR, refers to a molecule that promotes the associationof a first switch domain with a second switch domain. In embodiments,the dimerization molecule does not naturally occur in the subject, ordoes not occur in concentrations that would result in significantdimerization. In embodiments, the dimerization molecule is a smallmolecule, e.g., rapamycin or a rapalogue, e.g., RAD001.

The term “bioequivalent” refers to an amount of an agent other than thereference compound (e.g., RAD001), required to produce an effectequivalent to the effect produced by the reference dose or referenceamount of the reference compound (e.g., RAD001). In an embodiment theeffect is the level of mTOR inhibition, e.g., as measured by P70 S6kinase inhibition, e.g., as evaluated in an in vivo or in vitro assay,e.g., as measured by an assay described herein, e.g., the Boulay assay,or measurement of phosphorylated S6 levels by western blot. In anembodiment, the effect is alteration of the ratio of PD-1 positive/PD-1negative immune effector cells, e.g., T cells or NK cells, as measuredby cell sorting. In an embodiment a bioequivalent amount or dose of anmTOR inhibitor is the amount or dose that achieves the same level of P70S6 kinase inhibition as does the reference dose or reference amount of areference compound. In an embodiment, a bioequivalent amount or dose ofan mTOR inhibitor is the amount or dose that achieves the same level ofalteration in the ratio of PD-1 positive/PD-1 negative immune effectorcells, e.g., T cells or NK cells as does the reference dose or referenceamount of a reference compound.

The term “low, immune enhancing, dose” when used in conjunction with anmTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 orrapamycin, or a catalytic mTOR inhibitor, refers to a dose of mTORinhibitor that partially, but not fully, inhibits mTOR activity, e.g.,as measured by the inhibition of P70 S6 kinase activity. Methods forevaluating mTOR activity, e.g., by inhibition of P70 S6 kinase, arediscussed herein. The dose is insufficient to result in complete immunesuppression but is sufficient to enhance the immune response. In anembodiment, the low, immune enhancing, dose of mTOR inhibitor results ina decrease in the number of PD-1 positive immune effector cells, e.g., Tcells or NK cells and/or an increase in the number of PD-1 negativeimmune effector cells, e.g., T cells or NK cells, or an increase in theratio of PD-1 negative immune effector cells (e.g., T cells or NKcells)/PD-1 positive immune effector cells (e.g., T cells or NK cells).

In an embodiment, the low, immune enhancing, dose of mTOR inhibitorresults in an increase in the number of naive T cells. In an embodiment,the low, immune enhancing, dose of mTOR inhibitor results in one or moreof the following:

an increase in the expression of one or more of the following markers:CD62L^(high), CD127^(high), CD27⁺, and BCL2, e.g., on memory T cells,e.g., memory T cell precursors;

a decrease in the expression of KLRG1, e.g., on memory T cells, e.g.,memory T cell precursors; and

an increase in the number of memory T cell precursors, e.g., cells withany one or combination of the following characteristics: increasedCD62L^(high), increased CD127^(high), increased CD27⁺, decreased KLRG1,and increased BCL2;

wherein any of the changes described above occurs, e.g., at leasttransiently, e.g., as compared to a non-treated subject.

“Refractory” as used herein refers to a disease, e.g., cancer, that doesnot respond to a treatment. In embodiments, a refractory cancer can beresistant to a treatment before or at the beginning of the treatment. Inother embodiments, the refractory cancer can become resistant during atreatment. A refractory cancer is also called a resistant cancer.

“Relapsed” or a “relapse” as used herein refers to the reappearance of adisease (e.g., cancer) or the signs and symptoms of a disease such ascancer after a period of improvement or responsiveness, e.g., afterprior treatment of a therapy, e.g., cancer therapy. For example, theperiod of responsiveness may involve the level of cancer cells fallingbelow a certain threshold, e.g., below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or1%. The reappearance may involve the level of cancer cells rising abovea certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Asanother example, a range such as 95-99% identity, includes somethingwith 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This appliesregardless of the breadth of the range.

Description

Provided herein are compositions of matter and methods of use for thetreatment or prevention of a disease such as cancer using CD33 chimericantigen receptors (CAR).

In one aspect, the invention provides a number of chimeric antigenreceptors (CAR) comprising an antibody or antibody fragment engineeredfor specific binding to a CD33 protein or fragments thereof. In oneaspect, the invention provides a cell (e.g., an immune effector cell,e.g., T cell or NK cell) engineered to express a CAR, wherein the cell(e.g., “CART”) exhibits an antitumor property. In one aspect a cell istransformed with the CAR and the or at least part of the CAR isexpressed on the cell surface. In some embodiments, the cell (e.g.,immune effector cell, e.g., T cell or NK cell) is transduced with aviral vector encoding a CAR. In some embodiments, the viral vector is aretroviral vector. In some embodiments, the viral vector is a lentiviralvector. In some such embodiments, the cell may stably express the CAR.In another embodiment, the cell (e.g., immune effector cell, e.g., Tcell or NK cell) is transfected with a nucleic acid, e.g., mRNA, cDNA,DNA, encoding a CAR. In some such embodiments, the cell may transientlyexpress the CAR.

In one aspect, the CD33 binding domain, e.g., the human or humanizedCD33 binding domain, of the CAR is a scFv antibody fragment. In oneaspect, such antibody fragments are functional in that they retain theequivalent binding affinity, e.g., they bind the same antigen withcomparable efficacy, as the IgG antibody having the same heavy and lightchain variable regions. In one aspect such antibody fragments arefunctional in that they provide a biological response that can include,but is not limited to, activation of an immune response, inhibition ofsignal-transduction origination from its target antigen, inhibition ofkinase activity, and the like, as will be understood by a skilledartisan.

In some aspects, the antibodies of the invention are incorporated into achimeric antigen receptor (CAR). In one aspect, the CAR comprises thepolypeptide sequence provided herein as SEQ ID NOS: 48-56.

In one aspect, the CD33 binding domain, e.g., humanized or human CD33binding domain, portion of a CAR of the invention is encoded by atransgene whose sequence has been codon optimized for expression in amammalian cell. In one aspect, entire CAR construct of the invention isencoded by a transgene whose entire sequence has been codon optimizedfor expression in a mammalian cell. Codon optimization refers to thediscovery that the frequency of occurrence of synonymous codons (i.e.,codons that code for the same amino acid) in coding DNA is biased indifferent species. Such codon degeneracy allows an identical polypeptideto be encoded by a variety of nucleotide sequences. A variety of codonoptimization methods is known in the art, and include, e.g., methodsdisclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148.

In one aspect, the human CD33 binding domain comprises the scFv portionprovided in SEQ ID NO:39-47. In one aspect, the human CD33 bindingdomain comprises the scFv portion provided in SEQ ID NO: 39. In oneaspect, the human CD33 binding domain comprises the scFv portionprovided in SEQ ID NO: 40. In one aspect, the human CD33 binding domaincomprises the scFv portion provided in SEQ ID NO: 41. In one aspect, thehuman CD33 binding domain comprises the scFv portion provided in SEQ IDNO: 42. In one aspect, the human CD33 binding domain comprises the scFvportion provided in SEQ ID NO: 43. In one aspect, the human CD33 bindingdomain comprises the scFv portion provided in SEQ ID NO: 44. In oneaspect, the human CD33 binding domain comprises the scFv portionprovided in SEQ ID NO: 45. In one aspect, the human CD33 binding domaincomprises the scFv portion provided in SEQ ID NO: 46. In one aspect, thehuman CD33 binding domain comprises the scFv portion provided in SEQ IDNO: 47. In one aspect, the CARs of the invention combine an antigenbinding domain of a specific antibody with an intracellular signalingmolecule. For example, in some aspects, the intracellular signalingmolecule includes, but is not limited to, CD3-zeta chain, 4-1BB and CD28signaling modules and combinations thereof. In one aspect, the antigenbinding domain binds to CD33. In one aspect, the CD33 CAR comprises aCAR selected from the sequence provided in one or more of SEQ ID NOS:48-56. In one aspect, the CD33 CAR comprises the sequence provided inSEQ ID NO: 48. In one aspect, the CD33 CAR comprises the sequenceprovided in SEQ ID NO: 49. In one aspect, the CD33 CAR comprises thesequence provided in SEQ ID NO: 50. In one aspect, the CD33 CARcomprises the sequence provided in SEQ ID NO: 51. In one aspect, theCD33 CAR comprises the sequence provided in SEQ ID NO: 52. In oneaspect, the CD33 CAR comprises the sequence provided in SEQ ID NO: 53.In one aspect, the CD33 CAR comprises the sequence provided in SEQ IDNO: 54. In one aspect, the CD33 CAR comprises the sequence provided inSEQ ID NO: 55. In one aspect, the CD33 CAR comprises the sequenceprovided in SEQ ID NO: 56.

Furthermore, the present invention provides CD33 CAR compositions andtheir use in medicaments or methods for treating, among other diseases,cancer or any malignancy or autoimmune diseases involving cells ortissues which express CD33.

In one aspect, the CAR of the invention can be used to eradicateCD33-expressing normal cells, thereby applicable for use as a cellularconditioning therapy prior to cell transplantation or other suitabletherapy. Cell transplantation includes stem cell transplantation, e.g.,hematopoietic stem cell transplantation, and bone marrowtransplantation. The cell transplantation is allogeneic or autologous.In embodiments, the CAR of the invention eradicates CD33-expressingnormal cells or CD33-expressing cancer cells, or both, prior to celltransplantation or other suitable therapy. In one aspect, theCD33-expressing normal cell is a CD33-expressing expressing myeloidprogenitor cell and the cell transplantation is a stem celltransplantation.

In one aspect, the invention provides a cell (e.g., immune effectorcell, e.g., T cell or NK cell) engineered to express a chimeric antigenreceptor (e.g., immune effector cell, e.g., T cell or NK cell, e.g.,CART) of the present invention, wherein the cell (e.g., immune effectorcell, e.g., T cell or NK cell, e.g., “CART”) exhibits an antitumorproperty. Accordingly, the invention provides a CD33-CAR that comprisesa CD33 binding domain and is engineered into a cell (e.g., an immuneeffector cell, e.g., T cell or NK cell) and methods of their use foradoptive therapy.

In one aspect, the antigen binding domain of the CAR comprises a humanCD33 antibody or antibody fragment. In one aspect, the antigen bindingdomain of the CAR comprises a humanized CD33 antibody or antibodyfragment. In one aspect, the antigen binding domain of the CAR compriseshuman CD33 antibody fragment comprising an scFv. In one aspect, theantigen binding domain of the CAR is a human CD33 scFv. In one aspect,the antigen binding domain of the CAR comprises a humanized CD33antibody fragment comprising an scFv. In one aspect, the antigen bindingdomain of the CAR is a humanized CD33 scFv.

In one aspect, the CD33-CAR comprises at least one intracellular domain,e.g., described herein, e.g., selected from the group of a CD137 (4-1BB)signaling domain, a CD28 signaling domain, a CD3zeta signal domain, andany combination thereof. In one aspect, the CD33-CAR comprises at leastone intracellular signaling domain is from one or more co-stimulatorymolecule(s) other than a CD137 (4-1BB) or CD28.

Chimeric Antigen Receptor (CAR)

The present invention also provides a CAR (e.g., a CAR polypeptide) thatcomprises an anti-CD33 binding domain (e.g., a CD33 binding domain asdescribed herein), a transmembrane domain, and an intracellularsignaling domain, and wherein said CD33 binding domain comprises a heavychain complementary determining region 1 (HC CDR1), a heavy chaincomplementary determining region 2 (HC CDR2), and a heavy chaincomplementary determining region 3 (HC CDR3) of any heavy chain bindingdomain amino acid sequences listed in Table 2 or 9. The CD33 bindingdomain of the CAR can further comprise a light chain complementarydetermining region 1 (LC CDR1), a light chain complementary determiningregion 2 (LC CDR2), and a light chain complementary determining region 3(LC CDR3) of any heavy chain binding domain amino acid sequences listedin Table 2 or 9.

The present invention also provides nucleic acid molecules encoding theCAR as described herein, e.g., encoding a CAR that comprises a CD33binding domain (e.g., as described herein), a transmembrane domain, andan intracellular signaling domain, and wherein said CD33 binding domaincomprises a heavy chain complementary determining region 1 (HC CDR1), aheavy chain complementary determining region 2 (HC CDR2), and a heavychain complementary determining region 3 (HC CDR3) of any heavy chainbinding domain amino acid sequences listed in Table 2 or 9. In oneembodiment, the encoded CD33 binding domain of the CAR can furthercomprise a light chain complementary determining region 1 (LC CDR1), alight chain complementary determining region 2 (LC CDR2), and a lightchain complementary determining region 3 (LC CDR3) of any anti-BMCAheavy chain binding domain amino acid sequences listed in Table 2 or 9.

In specific aspects, a CAR construct of the invention comprises a humanscFv domain selected from the group consisting of SEQ ID NOS:39-47,wherein the scFv may be preceded by an optional leader sequence such asprovided in SEQ ID NO: 1, and followed by an optional hinge sequencesuch as provided in SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ IDNO:5, a transmembrane region such as provided in SEQ ID NO:6, anintracellular signalling domain that includes SEQ ID NO:7 or SEQ ID NO:8and a CD3 zeta sequence that includes SEQ ID NO:9 or SEQ ID NO:10, e.g.,wherein the domains are contiguous with and in the same reading frame toform a single fusion protein. Also included in the invention is anucleotide sequence that encodes the polypeptide of each of the humanscFv fragments selected from the group consisting of SEQ ID NO:39-47.Also included in the invention is a nucleotide sequence that encodes thepolypeptide of each of the human scFv fragments selected from the groupconsisting of SEQ ID NO: 39-47, and each of the domains of SEQ ID NOS:1,2, and 6-9, plus the encoded CD33 CAR of the invention.

In one aspect, an exemplary CD33CAR construct comprises an optionalleader sequence, an extracellular antigen binding domain, a hinge, atransmembrane domain, and an intracellular stimulatory domain. In oneaspect, an exemplary CD33CAR construct comprises an optional leadersequence, an extracellular antigen binding domain, a hinge, atransmembrane domain, an intracellular costimulatory domain and anintracellular stimulatory domain. Specific CD33 CAR constructscontaining humanized scFv domains of the invention are provided as SEQID NO: 138.

In some embodiments, full-length CD33 CAR sequences are also providedherein as SEQ ID NOS: 48-56, as shown in Table 2.

An exemplary leader sequence is provided as SEQ ID NO: 1. An exemplaryhinge/spacer sequence is provided as SEQ ID NO:2 or SEQ ID NO:3 or SEQID NO:4 or SEQ ID NO:5. An exemplary transmembrane domain sequence isprovided as SEQ ID NO:6. An exemplary sequence of the intracellularsignaling domain of the 4-1BB protein is provided as SEQ ID NO: 7. Anexemplary sequence of the intracellular signaling domain of CD27 isprovided as SEQ ID NO:8. An exemplary CD3zeta domain sequence isprovided as SEQ ID NO: 9 or SEQ ID NO:10.

In one aspect, the present invention encompasses a recombinant nucleicacid construct comprising a nucleic acid molecule encoding a CAR,wherein the nucleic acid molecule comprises the nucleic acid sequenceencoding a CD33 binding domain, e.g., described herein, e.g., that iscontiguous with and in the same reading frame as a nucleic acid sequenceencoding an intracellular signaling domain. In one aspect, a human CD33binding domain is selected from one or more of SEQ ID NOS:39-47. In oneaspect, the human CD33 binding domain is SEQ ID NO: 39. In one aspect,the human CD33 binding domain is SEQ ID NO: 40. In one aspect, the humanCD33 binding domain is SEQ ID NO: 41. In one aspect, the human CD33binding domain is SEQ ID NO: 42. In one aspect, the human CD33 bindingdomain is SEQ ID NO: 43. In one aspect, the human CD33 binding domain isSEQ ID NO: 44. In one aspect, the human CD33 binding domain is SEQ IDNO: 45. In one aspect, the human CD33 binding domain is SEQ ID NO: 46.In one aspect, the human CD33 binding domain is SEQ ID NO: 47.

In one aspect, the present invention encompasses a recombinant nucleicacid construct comprising a nucleic acid molecule encoding a CAR,wherein the nucleic acid molecule comprises a nucleic acid sequenceencoding a CD33 binding domain, e.g., wherein the sequence is contiguouswith and in the same reading frame as the nucleic acid sequence encodingan intracellular signaling domain. An exemplary intracellular signalingdomain that can be used in the CAR includes, but is not limited to, oneor more intracellular signaling domains of, e.g., CD3-zeta, CD28, 4-1BB,and the like. In some instances, the CAR can comprise any combination ofCD3-zeta, CD28, 4-1BB, and the like.

In one aspect, the nucleic acid sequence of a CAR construct of theinvention is selected from one or more of SEQ ID NOS:75-83. In oneaspect, the nucleic acid sequence of a CAR construct comprises (e.g.,consists of) SEQ ID NO:75. In one aspect, the nucleic acid sequence of aCAR construct comprises (e.g., consists of) SEQ ID NO:76. In one aspect,the nucleic acid sequence of a CAR construct comprises (e.g., consistsof) SEQ ID NO:77. In one aspect, the nucleic acid sequence of a CARconstruct comprises (e.g., consists of) SEQ ID NO:78. In one aspect, thenucleic acid sequence of a CAR construct comprises (e.g., consists of)SEQ ID NO:79. In one aspect, the nucleic acid sequence of a CARconstruct comprises (e.g., consists of) SEQ ID NO:80. In one aspect, thenucleic acid sequence of a CAR construct comprises (e.g., consists of)SEQ ID NO:81. In one aspect, the nucleic acid sequence of a CARconstruct comprises (e.g., consists of) SEQ ID NO:82. In one aspect, thenucleic acid sequence of a CAR construct comprises (e.g., consists of)SEQ ID NO:83.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the nucleic acid of interest can beproduced synthetically, rather than cloned. The present inventionincludes retroviral and lentiviral vector constructs expressing a CARthat can be directly transduced into a cell.

The present invention also includes an RNA construct that can bedirectly transfected into a cell. A method for generating mRNA for usein transfection involves in vitro transcription (IVT) of a template withspecially designed primers, followed by polyA addition, to produce aconstruct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ capand/or Internal Ribosome Entry Site (IRES), the nucleic acid to beexpressed, and a polyA tail, typically 50-2000 bases in length (SEQ IDNO:35). RNA so produced can efficiently transfect different kinds ofcells. In one embodiment, the template includes sequences for the CAR.In an embodiment, an RNA CAR vector is transduced into a cell byelectroporation.

Antigen Binding Domain

The CARs of the present invention comprise a target-specific bindingelement. The choice of moiety depends upon the type and number ofligands that define the surface of a target cell. For example, theantigen binding domain may be chosen to recognize an antigen that actsas a cell surface marker on target cells associated with a particulardisease state.

In one aspect, the CAR-mediated T-cell response can be directed to anantigen of interest by way of engineering an antigen binding domain thatspecifically binds a desired antigen into the CAR.

In one aspect, the CAR of the present invention comprises a bindingdomain that specifically binds to CD33. In one aspect, the CAR of thepresent invention comprises an antigen binding domain specifically bindsto human CD33.

The antigen binding domain can be any protein that binds to the antigenincluding but not limited to a monoclonal antibody, a polyclonalantibody, a recombinant antibody, a human antibody, a humanizedantibody, and a functional fragment thereof, including but not limitedto a single-domain antibody such as a heavy chain variable domain (VH),a light chain variable domain (VL) and a variable domain (VHH) ofcamelid derived nanobody, and to an alternative scaffold known in theart to function as antigen binding domain, such as a recombinantfibronectin domain, and the like. In some instances, it is beneficialfor the antigen binding domain to be derived from the same species inwhich the CAR will ultimately be used in. For example, for use inhumans, it may be beneficial for the antigen binding domain of the CARto comprise human or humanized residues for the antigen binding domainof an antibody or antibody fragment.

In some instances, it is beneficial for the antigen binding domain to bederived from the same species in which the CAR will ultimately be usedin. For example, for use in humans, it may be beneficial for the antigenbinding domain of the CAR to comprise human or humanized residues forthe antigen binding domain of an antibody or antibody fragment. Thus, inone aspect, the antigen binding domain comprises a human antibody or anantibody fragment. In one embodiment, the human CD33 binding domaincomprises one or more (e.g., all three) light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) of a human CD33 binding domain described herein, and/or one ormore (e.g., all three) heavy chain complementary determining region 1(HC CDR1), heavy chain complementary determining region 2 (HC CDR2), andheavy chain complementary determining region 3 (HC CDR3) of a human CD33binding domain described herein, e.g., a human CD33 binding domaincomprising one or more, e.g., all three, LC CDRs and one or more, e.g.,all three, HC CDRs. In one embodiment, the human CD33 binding domaincomprises one or more (e.g., all three) heavy chain complementarydetermining region 1 (HC CDR1), heavy chain complementary determiningregion 2 (HC CDR2), and heavy chain complementary determining region 3(HC CDR3) of a human CD33 binding domain described herein, e.g., thehuman CD33 binding domain has two variable heavy chain regions, eachcomprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein. In oneembodiment, the human CD33 binding domain comprises a human light chainvariable region described herein (e.g., in Table 4) and/or a human heavychain variable region described herein (e.g., in Table 3). In oneembodiment, the human CD33 binding domain comprises a human heavy chainvariable region described herein (e.g., in Table 3), e.g., at least twohuman heavy chain variable regions described herein (e.g., in Table 3).In one embodiment, the CD33 binding domain is a scFv comprising a lightchain and a heavy chain of an amino acid sequence of Tables 3-4. In anembodiment, the CD33 binding domain (e.g., an scFv) comprises: a lightchain variable region comprising an amino acid sequence having at leastone, two or three modifications (e.g., substitutions, e.g., conservativesubstitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions, e.g., conservative substitutions) of an amino acidsequence of a light chain variable region provided in Table 4, or asequence with 95-99% identity with an amino acid sequence of Table 5;and/or a heavy chain variable region comprising an amino acid sequencehaving at least one, two or three modifications (e.g., substitutions,e.g., conservative substitutions) but not more than 30, 20 or 10modifications (e.g., substitutions, e.g., conservative substitutions) ofan amino acid sequence of a heavy chain variable region provided inTable 4, or a sequence with 95-99% identity to an amino acid sequence ofTable 3. In one embodiment, the human CD33 binding domain comprises asequence selected from a group consisting of SEQ ID NO:39-47, or asequence with 95-99% identity thereof. CD33 In one embodiment, the humanCD33 binding domain is a scFv, and a light chain variable regioncomprising an amino acid sequence described herein, e.g., in Table 4, isattached to a heavy chain variable region comprising an amino acidsequence described herein, e.g., in Table 3, via a linker, e.g., alinker described herein. In one embodiment, the human CD33 bindingdomain includes a (Gly₄-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6,preferably 3 or 4 (SEQ ID NO:26). The light chain variable region andheavy chain variable region of a scFv can be, e.g., in any of thefollowing orientations: light chain variable region-linker-heavy chainvariable region or heavy chain variable region-linker-light chainvariable region.

In one aspect, the antigen binding domain comprises a humanized antibodyor an antibody fragment. In one embodiment, the humanized CD33 bindingdomain comprises one or more (e.g., all three) light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) of a CD33 binding domain described herein, and/or one or more(e.g., all three) heavy chain complementary determining region 1 (HCCDR1), heavy chain complementary determining region 2 (HC CDR2), andheavy chain complementary determining region 3 (HC CDR3) of a CD33binding domain described herein, e.g., a humanized CD33 binding domaincomprising one or more, e.g., all three, LC CDRs and one or more, e.g.,all three, HC CDRs. In one embodiment, the humanized CD33 binding domainincludes a (Gly₄-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6,preferably 3 or 4 (SEQ ID NO:26). The light chain variable region andheavy chain variable region of a scFv can be, e.g., in any of thefollowing orientations: light chain variable region-linker-heavy chainvariable region or heavy chain variable region-linker-light chainvariable region.

In one aspect, the CD33 CAR that includes a humanized CD33 bindingdomain comprises SEQ ID NOS: 143. In some aspects, a non-human antibodyis humanized, where specific sequences or regions of the antibody aremodified to increase similarity to an antibody naturally produced in ahuman or fragment thereof. In one aspect, the antigen binding domain ishumanized. Examples of humanized CD33 antibodies for use with CARTdescribed herein include hp67.6 or Gemtuzumab, as described inWO2012123755.

A humanized antibody can be produced using a variety of techniques knownin the art, including but not limited to, CDR-grafting (see, e.g.,European Patent No. EP 239,400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, eachof which is incorporated herein in its entirety by reference), veneeringor resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al.,1994, PNAS, 91:969-973, each of which is incorporated herein by itsentirety by reference), chain shuffling (see, e.g., U.S. Pat. No.5,565,332, which is incorporated herein in its entirety by reference),and techniques disclosed in, e.g., U.S. Patent Application PublicationNo. US2005/0042664, U.S. Patent Application Publication No.US2005/0048617, U.S. Pat. Nos. 6,407,213, 5,766,886, InternationalPublication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002),Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods,20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84(1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto etal., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., CancerRes., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), andPedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which isincorporated herein in its entirety by reference. Often, frameworkresidues in the framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, for exampleimprove, antigen binding. These framework substitutions are identifiedby methods well-known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature,332:323, which are incorporated herein by reference in theirentireties.)

A humanized antibody or antibody fragment has one or more amino acidresidues remaining in it from a source which is nonhuman. These nonhumanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. As providedherein, humanized antibodies or antibody fragments comprise one or moreCDRs from nonhuman immunoglobulin molecules and framework regionswherein the amino acid residues comprising the framework are derivedcompletely or mostly from human germline. Multiple techniques forhumanization of antibodies or antibody fragments are well-known in theart and can essentially be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody, i.e., CDR-grafting (EP239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567;6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents ofwhich are incorporated herein by reference herein in their entirety). Insuch humanized antibodies and antibody fragments, substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a nonhuman species. Humanized antibodies areoften human antibodies in which some CDR residues and possibly someframework (FR) residues are substituted by residues from analogous sitesin rodent antibodies. Humanization of antibodies and antibody fragmentscan also be achieved by veneering or resurfacing (EP 592,106; EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al.,PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332),the contents of which are incorporated herein by reference herein intheir entirety.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is to reduce antigenicity. Accordingto the so-called “best-fit” method, the sequence of the variable domainof a rodent antibody is screened against the entire library of knownhuman variable-domain sequences. The human sequence which is closest tothat of the rodent is then accepted as the human framework (FR) for thehumanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothiaet al., J. Mol. Biol., 196:901 (1987), the contents of which areincorporated herein by reference herein in their entirety). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (see, e.g., Nicholson et al. Mol. Immun. 34 (16-17):1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993), the contents ofwhich are incorporated herein by reference herein in their entirety). Insome embodiments, the framework region, e.g., all four frameworkregions, of the heavy chain variable region are derived from a VH4_4-59germline sequence. In one embodiment, the framework region can comprise,one, two, three, four or five modifications, e.g., substitutions, e.g.,from the amino acid at the corresponding murine sequence (e.g., of SEQID NO:138). In one embodiment, the framework region, e.g., all fourframework regions of the light chain variable region are derived from aVK3_1.25 germline sequence. In one embodiment, the framework region cancomprise, one, two, three, four or five modifications, e.g.,substitutions, e.g., from the amino acid at the corresponding murinesequence (e.g., of SEQ ID NO:138).

In some aspects, the portion of a CAR composition of the invention thatcomprises an antibody fragment is humanized with retention of highaffinity for the target antigen and other favorable biologicalproperties. According to one aspect of the invention, humanizedantibodies and antibody fragments are prepared by a process of analysisof the parental sequences and various conceptual humanized productsusing three-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, e.g., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind the target antigen. In this way, FR residues canbe selected and combined from the recipient and import sequences so thatthe desired antibody or antibody fragment characteristic, such asincreased affinity for the target antigen, is achieved. In general, theCDR residues are directly and most substantially involved in influencingantigen binding.

A humanized antibody or antibody fragment may retain a similar antigenicspecificity as the original antibody, e.g., in the present invention,the ability to bind human CD33 or a fragment thereof. In someembodiments, a humanized antibody or antibody fragment may have improvedaffinity and/or specificity of binding to human CD33 or a fragmentthereof.

In one aspect, the antigen binding domain portion comprises one or moresequence selected from SEQ ID NOs:39-47. In one aspect, the CD33 CARthat includes a human CD33 binding domain is selected from one or moresequence selected from SEQ ID NOs: 48-56.

In one aspect, the CD33 binding domain is characterized by particularfunctional features or properties of an antibody or antibody fragment.For example, in one aspect, the portion of a CAR composition of theinvention that comprises an antigen binding domain specifically bindshuman CD33 or a fragment thereof. In one aspect, the invention relatesto an antigen binding domain comprising an antibody or antibodyfragment, wherein the antibody binding domain specifically binds to aCD33 protein or fragment thereof, wherein the antibody or antibodyfragment comprises a variable light chain and/or a variable heavy chainthat includes an amino acid sequence of SEQ ID NO: 48-56. In one aspect,the antigen binding domain comprises an amino acid sequence of an scFvselected from SEQ ID NO: 39-47. In certain aspects, the scFv iscontiguous with and in the same reading frame as a leader sequence. Inone aspect the leader sequence is the polypeptide sequence provided asSEQ ID NO:1.

In one aspect, the CD33 binding domain is a fragment, e.g., a singlechain variable fragment (scFv). In one aspect, the CD33 binding domainis a Fv, a Fab, a (Fab)2, or a bi-functional (e.g. bi-specific) hybridantibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)).In one aspect, the antibodies and fragments thereof of the inventionbinds a CD33 protein or a fragment thereof with wild-type or enhancedaffinity.

In some instances, a human scFv can be derived from a display library. Adisplay library is a collection of entities; each entity includes anaccessible polypeptide component and a recoverable component thatencodes or identifies the polypeptide component. The polypeptidecomponent is varied so that different amino acid sequences arerepresented. The polypeptide component can be of any length, e.g. fromthree amino acids to over 300 amino acids. A display library entity caninclude more than one polypeptide component, for example, the twopolypeptide chains of a Fab. In one exemplary embodiment, a displaylibrary can be used to identify a human CD33 binding domain. In aselection, the polypeptide component of each member of the library isprobed with CD33, or a fragment thereof, and if the polypeptidecomponent binds to CD33, the display library member is identified,typically by retention on a support.

Retained display library members are recovered from the support andanalyzed. The analysis can include amplification and a subsequentselection under similar or dissimilar conditions. For example, positiveand negative selections can be alternated. The analysis can also includedetermining the amino acid sequence of the polypeptide component, i.e.,the anti-CD33 binding domain, and purification of the polypeptidecomponent for detailed characterization.

A variety of formats can be used for display libraries. Examples includethe phaage display. In phage display, the protein component is typicallycovalently linked to a bacteriophage coat protein. The linkage resultsfrom translation of a nucleic acid encoding the protein component fusedto the coat protein. The linkage can include a flexible peptide linker,a protease site, or an amino acid incorporated as a result ofsuppression of a stop codon. Phage display is described, for example, inU.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; WO92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO92/01047; WO 92/09690; WO 90/02809; de Haard et al. (1999) J. Biol. Chem274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20;Hoogenboom et al. (2000) Immunol Today 2:371-8 and Hoet et al. (2005)Nat Biotechnol. 23(3)344-8. Bacteriophage displaying the proteincomponent can be grown and harvested using standard phage preparatorymethods, e.g. PEG precipitation from growth media. After selection ofindividual display phages, the nucleic acid encoding the selectedprotein components can be isolated from cells infected with the selectedphages or from the phage themselves, after amplification. Individualcolonies or plaques can be picked, the nucleic acid isolated andsequenced.

Other display formats include cell based display (see, e.g., WO03/029456), protein-nucleic acid fusions (see, e.g., U.S. Pat. No.6,207,446), ribosome display (See, e.g., Mattheakis et al. (1994) Proc.Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol.18:1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30; andSchaffitzel et al. (1999) J Immunol Methods. 231(1-2):119-35), and E.coli periplasmic display (2005 Nov. 22; PMID: 16337958).

In some instances, scFvs can be prepared according to method known inthe art (see, for example, Bird et al., (1988) Science 242:423-426 andHuston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFvmolecules can be produced by linking VH and VL regions together usingflexible polypeptide linkers. The scFv molecules comprise a linker(e.g., a Ser-Gly linker) with an optimized length and/or amino acidcomposition. The linker length can greatly affect how the variableregions of a scFv fold and interact. In fact, if a short polypeptidelinker is employed (e.g., between 5-10 amino acids) intrachain foldingis prevented. Interchain folding is also required to bring the twovariable regions together to form a functional epitope binding site. Forexamples of linker orientation and size see, e.g., Hollinger et al. 1993Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent ApplicationPublication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCTpublication Nos. WO2006/020258 and WO2007/024715, is incorporated hereinby reference.

An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or moreamino acid residues between its VL and VH regions. The linker sequencemay comprise any naturally occurring amino acid. In some embodiments,the linker sequence comprises amino acids glycine and serine. In anotherembodiment, the linker sequence comprises sets of glycine and serinerepeats such as (Gly₄Ser)n, where n is a positive integer equal to orgreater than 1 (SEQ ID NO:25). In one embodiment, the linker can be(Gly₄Ser)₄ (SEQ ID NO:27) or (Gly₄Ser)₃(SEQ ID NO:28). Variation in thelinker length may retain or enhance activity, giving rise to superiorefficacy in activity studies.

Exemplary Human CD33 CAR Constructs and Antigen Binding Domains

Exemplary CD33 CAR constructs disclose herein comprise an scFv (e.g., ahuman scFv as disclosed in Tables 2 and 9 herein, optionally precededwith an optional leader sequence (e.g., SEQ ID NO:1 and SEQ ID NO:12 forexemplary leader amino acid and nucleotide sequences, respectively). Thesequences of the human scFv fragments (SEQ ID NOs: 39-83, including theoptional leader sequence) are provided herein in Table 2. The sequencesof human scFv fragments, without the leader sequence, are providedherein in Table 9 (SEQ ID NOS: 255-261 for the nucleotide sequences, andSEQ ID NOs: 262-268 for the amino acid sequences). The CD33 CARconstruct can further include an optional hinge domain, e.g., a CD8hinge domain (e.g., including the amino acid sequence of SEQ ID NO: 2 orencoded by a nucleic acid sequence of SEQ ID NO:13); a transmembranedomain, e.g., a CD8 transmembrane domain (e.g., including the amino acidsequence of SEQ ID NO: 6 or encoded by the nucleotide sequence of SEQ IDNO: 17); an intracellular domain, e.g., a 4-1BB intracellular domain(e.g., including the amino acid sequence of SEQ ID NO: 7 or encoded bythe nucleotide sequence of SEQ ID NO: 18; and a functional signalingdomain, e.g., a CD3 zeta domain (e.g., including amino acid sequence ofSEQ ID NO: 9 or 10, or encoded by the nucleotide sequence of SEQ ID NO:20 or 21). In certain embodiments, the domains are contiguous with andin the same reading frame to form a single fusion protein. In otherembodiments, the domain are in separate polypeptides, e.g., as in anRCAR molecule as described herein.

In certain embodiments, the full length CD33 CAR molecule includes theamino acid sequence of, or is encoded by the nucleotide sequence of,CAR33-1, CAR33-2, CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8,CAR33-9, provided in Table 2, or a sequence substantially (e.g., 95-99%)identical thereto.

In certain embodiments, the CD33 CAR molecule, or the anti-CD33 antigenbinding domain, includes the scFv amino acid sequence of CAR33-1,CAR33-2, CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9,provided in Table 2 (with or without the leader sequence); or includesthe scFv amino acid sequence of, or is encoded by the nucleotidesequence of, CAR33-1, CAR33-2, CAR33-4, CAR33-5, CAR33-6, CAR33-7,CAR33-9, provided in Table 9, or a sequence substantially identical(e.g., 95-99% identical, or up to 20, 15, 10, 8, 6, 5, 4, 3, 2, or 1amino acid changes, e.g., substitutions (e.g., conservativesubstitutions)) to any of the aforesaid sequences.

In certain embodiments, the CD33 CAR molecule, or the anti-CD33 antigenbinding domain, includes the heavy chain variable region and/or thelight chain variable region of CAR33-1, CAR33-2, CAR33-3, CAR33-4,CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9, provided in Table 2, or asequence substantially identical (e.g., 95-99% identical, or up to 20,15, 10, 8, 6, 5, 4, 3, 2, or 1 amino acid changes, e.g., substitutions(e.g., conservative substitutions)) to any of the aforesaid sequences.

In certain embodiments, the CD33 CAR molecule, or the anti-CD33 antigenbinding domain, includes one, two or three CDRs from the heavy chainvariable region (e.g., HC CDR1, HC CDR2 and/or HC CDR3), provided inTable 3; and/or one, two or three CDRs from the light chain variableregion (e.g., LC CDR1, LC CDR2 and/or LC CDR3) of CAR33-1, CAR33-2,CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9, providedin Table 4; or a sequence substantially identical (e.g., 95-99%identical, or up to 5, 4, 3, 2, or 1 amino acid changes, e.g.,substitutions (e.g., conservative substitutions)) to any of theaforesaid sequences.

In certain embodiments, the CD33 CAR molecule, or the anti-CD33 antigenbinding domain, includes one, two or three CDRs from the heavy chainvariable region (e.g., HC CDR1, HC CDR2 and/or HC CDR3), provided inTable 10; and/or one, two or three CDRs from the light chain variableregion (e.g., LC CDR1, LC CDR2 and/or LC CDR3) of CAR33-1, CAR33-2,CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9, providedin Table 11; or a sequence substantially identical (e.g., 95-99%identical, or up to 5, 4, 3, 2, or 1 amino acid changes, e.g.,substitutions (e.g., conservative substitutions)) to any of theaforesaid sequences.

In certain embodiments, the CD33 CAR molecule, or the anti-CD33 antigenbinding domain, includes one, two or three CDRs from the heavy chainvariable region (e.g., HC CDR1, HC CDR2 and/or HC CDR3), provided inTable 12; and/or one, two or three CDRs from the light chain variableregion (e.g., LCDR1, LCDR2 and/or LCDR3) of CAR33-1, CAR33-2, CAR33-3,CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9, provided in Table13; or a sequence substantially identical (e.g., 95-99% identical, or upto 5, 4, 3, 2, or 1 amino acid changes, e.g., substitutions (e.g.,conservative substitutions)) to any of the aforesaid sequences.

The sequences of human CDR sequences of the scFv domains are shown inTable 3 for the heavy chain variable domains and in Table 4 for thelight chain variable domains. “ID” stands for the respective SEQ ID NOfor each CDR. The CDRs provided in Tables 3 and 4 are according to acombination of the Kabat and Chothia numbering scheme.

TABLE 3 Heavy Chain Variable Domain CDRs Candidate HCDR1 ID HCDR2 IDHCDR3 ID CAR33-1 GYSFTSYWIG 84 IIYPGDSDTRYSPSFQG 93 LGGSLPDYGMDV 102CAR33-2 GYIFTNYYVH 85 IISPSGGSPTYAQRLQG 94 ESRLRGNRLGLQSSIFDH 103CAR33-3 GFTFSSYAMS 86 AISGSGGSTYYADSVKG 95 EDTIRGPNYYYYGMDV 104 CAR33-4GYSFTSYWIG 87 IIYPGDSDTRYSPSFQG 96 GGYSDYDYYFDF 105 CAR33-5 GFTFDDYAMH88 VIWPDGGQKYYGDSVKG 97 HFNAWDY 106 CAR33-6 GFTFSIFAMH 89TISYDGSNAFYADSVEG 98 AGDGGYDVFDS 107 CAR33-7 GFTFSSYAMS 90AISGSGGSTYYADSVKG 99 ETDYYGSGTFDY 108 CAR33-8 GYMFTDFFIH 91WINPNSGVTKYAQKFQG 100 WYSSGWYGIANI 109 CAR33-9 GYSFTNYWIG 92IIYPGDSDTRYSPSFQG 101 HGPSSWGEFDY 110

TABLE 4 Light Chain Variable Domain CDRs Candidate LCDR1 ID LCDR2 IDLCDR3 ID CAR33-1 RSSQSLLHSNG 111 LGSNRAS 120 MQALQTLIT 129 YNYLD CAR33-2QASQDINNHLN 112 DTSNLEI 121 QQYENLPLT 130 CAR33-3 RASQDIDTWLA 113AASNLQG 122 QQASIFPPT 131 CAR33-4 RSSQSLLHSNG 114 LGSNRAS 123 MQALQTPFT132 YNYLD CAR33-5 QASQGISQFLN 115 DASNLEP 124 QQYDDLPLT 133 CAR33-6RSSQSLLHSNG 116 LGSNRAS 125 MQALQTPT 134 YNYLD CAR33-7 RASQGIGIYLA 117GASTLQS 126 QQSNNFPPT 135 CAR33-8 QASHDISNYLH 118 DASNLET 127 QQSDDLPHT136 CAR33-9 RASQSISSYLN 119 AASSLQS 128 QQSYSTPLT 137

TABLE 10 Heavy Chain Variable Domain CDRs according to the Kabat numbering scheme (Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service,National Institutes of Health, Bethesda, MD) Candidate HCDR1 ID HCDR2 IDHCDR3 ID 141643- SYWIG 269 IIYPGDSDTRYSPSFQG 278 LGGSLPDYGMDV 287CAR33-1 141644- NYYVH 270 IISPSGGSPTYAQRLQG 279 ESRLRGNRLGLQSSIFDH 288CAR33-2 141645- SYAMS 271 AISGSGGSTYYADSVKG 280 EDTIRGPNYYYYGMDV 289CAR33-3 141646- SYWIG 272 IIYPGDSDTRYSPSFQG 281 GGYSDYDYYFDF 290 CAR33-4141647- DYAMH 273 VIWPDGGQKYYGDSVKG 282 HFNAWDY 291 CAR33-5 141648-IFAMH 274 TISYDGSNAFYADSVEG 283 AGDGGYDVFDS 292 CAR33-6 141649- SYAMS275 AISGSGGSTYYADSVKG 284 ETDYYGSGTFDY 293 CAR33-7 141650- DFFIH 276WINPNSGVTKYAQKFQG 285 WYSSGWYGIANI 294 CAR33-8 141651- NYWIG 277IIYPGDSDTRYSPSFQG 286 HGPSSWGEFDY 295 CAR33-9

TABLE 11 Light Chain Variable Domain CDRs according to the Kabatnumbering scheme (Kabat et al. (1991), “Sequences of Proteinsof Immunological Interest,” 5th Ed. Public Health Service,National Institutes of Health, Bethesda, MD) Candidate LCDR1 ID LCDR2 IDLCDR3 ID 141643- RSSQSLLHSNGYNYLD 296 LGSNRAS 305 MQALQTLIT 314 CAR33-1141644- QASQDINNHLN 297 DTSNLEI 306 QQYENLPLT 315 CAR33-2 141645-RASQDIDTWLA 298 AASNLQG 307 QQASIFPPT 316 CAR33-3 141646-RSSQSLLHSNGYNYLD 299 LGSNRAS 308 MQALQTPFT 317 CAR33-4 141647-QASQGISQFLN 300 DASNLEP 309 QQYDDLPLT 318 CAR33-5 141648-RSSQSLLHSNGYNYLD 301 LGSNRAS 310 MQALQTPT 319 CAR33-6 141649-RASQGIGIYLA 302 GASTLQS 311 QQSNNFPPT 320 CAR33-7 141650- QASHDISNYLH303 DASNLET 312 QQSDDLPHT 321 CAR33-8 141651- RASQSISSYLN 304 AASSLQS313 QQSYSTPLT 322 CAR33-9

TABLE 12 Heavy Chain Variable Domain CDRsaccording to the Chothia numbering scheme(Al-Lazikani et al., (1997) JMB 273,927-948) Candidate HCDR1 ID HCDR2 IDHCDR3 ID 141643- GYSFTSY 323 YPGDSD 332 LGGSLPDYGMDV 341 CAR33-1 141644-GYIFTNY 324 SPSGGS 333 ESRLRGNRLGLQ 342 CAR33-2 SSIFDH 141645- GFTFSSY325 SGSGGS 334 EDTIRGPNYYYY 343 CAR33-3 GMDV 141646- GYSFTSY 326 YPGDSD335 GGYSDYDYYFDF 344 CAR33-4 141647- GFTFDDY 327 WPDGGQ 336 HFNAWDY 345CAR33-5 141648- GFTFSIF 328 SYDGSN 337 AGDGGYDVFDS 346 CAR33-6 141649-GFTFSSY 329 SGSGGS 338 ETDYYGSGTFDY 347 CAR33-7 141650- GYMFTDF 330NPNSGV 339 WYSSGWYGIANI 348 CAR33-8 141651- GYSFTNY 331 YPGDSD 340HGPSSWGEFDY 349 CAR33-9

TABLE 13 Light Chain Variable Domain CDRsaccording to the Chothia numbering scheme(Al-Lazikani et al., (1997) JMB 273,927-948) Candidate LCDR1 ID LCDR2 IDLCDR3 ID 141643- SQSLLHSNGYNY 350 LGS 359 ALQTLI 368 CAR33-1 141644-SQDINNH 351 DTS 360 YENLPL 369 CAR33-2 141645- SQDIDTW 352 AAS 361ASIFPP 370 CAR33-3 141646- SQSLLHSNGYNY 353 LGS 362 ALQTPF 371 CAR33-4141647- SQGISQF 354 DAS 363 YDDLPL 372 CAR33-5 141648- SQSLLHSNGYNY 355LGS 364 ALQTP 373 CAR33-6 141649- SQGIGIY 356 GAS 365 SNNFPP 374 CAR33-7141650- SHDISNY 357 DAS 366 SDDLPH 375 CAR33-8 141651- SQSISSY 358 AAS367 SYSTPL 376 CAR33-9

In certain embodiments, the CAR molecule described herein (e.g., the CARnucleic acid or the CAR polypeptide) or a CD33 binding domain includes:

(1) one, two, or three light chain (LC) CDRs chosen from one of thefollowing:

(i) a LC CDR1 of SEQ ID NO: 111, LC CDR2 of SEQ ID NO: 120 and LC CDR3of SEQ ID NO: 129 of CAR33-1;

(ii) a LC CDR1 of SEQ ID NO: 112, LC CDR2 of SEQ ID NO: 121 and LC CDR3of SEQ ID NO: 130 of CAR33-2;

(iii) a LC CDR1 of SEQ ID NO: 113, LC CDR2 of SEQ ID NO: 122 and LC CDR3of SEQ ID NO: 131 of CAR33-3;

(iv) a LC CDR1 of SEQ ID NO: 114, LC CDR2 of SEQ ID NO: 123 and LC CDR3of SEQ ID NO: 132 of CAR33-4;

(iv) a LC CDR1 of SEQ ID NO: 115, LC CDR2 of SEQ ID NO: 124 and LC CDR3of SEQ ID NO: 133 of CAR33-5;

(vi) a LC CDR1 of SEQ ID NO: 116, LC CDR2 of SEQ ID NO: 125 and LC CDR3of SEQ ID NO: 134 of CAR33-6;

(vii) a LC CDR1 of SEQ ID NO: 117, LC CDR2 of SEQ ID NO: 126 and LC CDR3of SEQ ID NO: 135 of CAR33-7;

(viii) a LC CDR1 of SEQ ID NO: 118, LC CDR2 of SEQ ID NO: 127 and LCCDR3 of SEQ ID NO: 136 of CAR33-8; or

(ix) a LC CDR1 of SEQ ID NO: 119, LC CDR2 of SEQ ID NO: 128 and LC CDR3of SEQ ID NO: 137 of CAR33-9; and/or

(2) one, two, or three heavy chain (HC) CDRs from one of the following:

(i) a HC CDR1 of SEQ ID NO: 84, HC CDR2 of SEQ ID NO: 93 and HC CDR3 ofSEQ ID NO: 102 of CAR33-1;

(ii) a HC CDR1 of SEQ ID NO: 85, HC CDR2 of SEQ ID NO: 94 and HC CDR3 ofSEQ ID NO: 103 of CAR33-2;

(iii) a HC CDR1 of SEQ ID NO: 86, HC CDR2 of SEQ ID NO: 95 and HC CDR3of SEQ ID NO: 104 of CAR33-3;

(iv) a HC CDR1 of SEQ ID NO: 87, HC CDR2 of SEQ ID NO: 96 and HC CDR3 ofSEQ ID NO: 105 of CAR33-4;

(iv) a HC CDR1 of SEQ ID NO: 88, HC CDR2 of SEQ ID NO: 97 and HC CDR3 ofSEQ ID NO: 106 of CAR33-5;

(vi) a HC CDR1 of SEQ ID NO: 89, HC CDR2 of SEQ ID NO: 98 and HC CDR3 ofSEQ ID NO: 107 of CAR33-6;

(vii) a HC CDR1 of SEQ ID NO: 90, HC CDR2 of SEQ ID NO: 99 and HC CDR3of SEQ ID NO: 108 of CAR33-7;

(viii) a HC CDR1 of SEQ ID NO: 91, HC CDR2 of SEQ ID NO: 100 and HC CDR3of SEQ ID NO: 109 of CAR33-8; or

(ix) a HC CDR1 of SEQ ID NO: 92, HC CDR2 of SEQ ID NO: 101 and HC CDR3of SEQ ID NO: 110 of CAR33-9.

In certain embodiments, the CAR molecule described herein (e.g., the CARnucleic acid or the CAR polypeptide) includes:

(1) one, two, or three light chain (LC) CDRs chosen from one of thefollowing:

(i) a LC CDR1 of SEQ ID NO: 296, LC CDR2 of SEQ ID NO: 305 and LC CDR3of SEQ ID NO: 314 of CAR33-1;

(ii) a LC CDR1 of SEQ ID NO: 297, LC CDR2 of SEQ ID NO: 306 and LC CDR3of SEQ ID NO: 315 of CAR33-2;

(iii) a LC CDR1 of SEQ ID NO: 298, LC CDR2 of SEQ ID NO: 307 and LC CDR3of SEQ ID NO: 316 of CAR33-3;

(iv) a LC CDR1 of SEQ ID NO: 299, LC CDR2 of SEQ ID NO: 308 and LC CDR3of SEQ ID NO: 317 of CAR33-4;

(iv) a LC CDR1 of SEQ ID NO: 300, LC CDR2 of SEQ ID NO: 309 and LC CDR3of SEQ ID NO: 318 of CAR33-5;

(vi) a LC CDR1 of SEQ ID NO: 301, LC CDR2 of SEQ ID NO: 310 and LC CDR3of SEQ ID NO: 319 of CAR33-6;

(vii) a LC CDR1 of SEQ ID NO: 302, LC CDR2 of SEQ ID NO: 311 and LC CDR3of SEQ ID NO: 320 of CAR33-7;

(viii) a LC CDR1 of SEQ ID NO: 303, LC CDR2 of SEQ ID NO: 312 and LCCDR3 of SEQ ID NO: 321 of CAR33-8; or

(ix) a LC CDR1 of SEQ ID NO: 304, LC CDR2 of SEQ ID NO: 313 and LC CDR3of SEQ ID NO: 322 of CAR33-9; and/or

(2) one, two, or three heavy chain (HC) CDRs chosen from one of thefollowing:

(i) a HC CDR1 of SEQ ID NO: 269, HC CDR2 of SEQ ID NO: 278 and HC CDR3of SEQ ID NO: 287 of CAR33-1;

(ii) a HC CDR1 of SEQ ID NO: 270, HC CDR2 of SEQ ID NO: 279 and HC CDR3of SEQ ID NO: 288 of CAR33-2;

(iii) a HC CDR1 of SEQ ID NO: 271, HC CDR2 of SEQ ID NO: 280 and HC CDR3of SEQ ID NO: 289 of CAR33-3;

(iv) a HC CDR1 of SEQ ID NO: 272, HC CDR2 of SEQ ID NO: 281 and HC CDR3of SEQ ID NO: 290 of CAR33-4;

(iv) a HC CDR1 of SEQ ID NO: 273, HC CDR2 of SEQ ID NO: 282 and HC CDR3of SEQ ID NO: 291 of CAR33-5;

(vi) a HC CDR1 of SEQ ID NO: 274, HC CDR2 of SEQ ID NO: 283 and HC CDR3of SEQ ID NO: 292 of CAR33-6;

(vii) a HC CDR1 of SEQ ID NO: 275, HC CDR2 of SEQ ID NO: 284 and HC CDR3of SEQ ID NO: 293 of CAR33-7;

(viii) a HC CDR1 of SEQ ID NO: 276, HC CDR2 of SEQ ID NO: 285 and HCCDR3 of SEQ ID NO: 294 of CAR33-8; or

(ix) a HC CDR1 of SEQ ID NO: 277, HC CDR2 of SEQ ID NO: 286 and HC CDR3of SEQ ID NO: 295 of CAR33-9.

In certain embodiments, the CAR molecule described herein (e.g., the CARnucleic acid or the CAR polypeptide) includes:

(1) one, two, or three light chain (LC) CDRs chosen from one of thefollowing:

(i) a LC CDR1 of SEQ ID NO: 350, LC CDR2 of SEQ ID NO: 359 and LC CDR3of SEQ ID NO: 368 of CAR33-1;

(ii) a LC CDR1 of SEQ ID NO: 351, LC CDR2 of SEQ ID NO: 360 and LC CDR3of SEQ ID NO: 369 of CAR33-2;

(iii) a LC CDR1 of SEQ ID NO: 352, LC CDR2 of SEQ ID NO: 361 and LC CDR3of SEQ ID NO: 370 of CAR33-3;

(iv) a LC CDR1 of SEQ ID NO: 353, LC CDR2 of SEQ ID NO: 362 and LC CDR3of SEQ ID NO: 371 of CAR33-4;

(iv) a LC CDR1 of SEQ ID NO: 354, LC CDR2 of SEQ ID NO: 363 and LC CDR3of SEQ ID NO: 372 of CAR33-5;

(vi) a LC CDR1 of SEQ ID NO: 355, LC CDR2 of SEQ ID NO: 364 and LC CDR3of SEQ ID NO: 373 of CAR33-6;

(vii) a LC CDR1 of SEQ ID NO: 356, LC CDR2 of SEQ ID NO: 365 and LC CDR3of SEQ ID NO: 374 of CAR33-7;

(viii) a LC CDR1 of SEQ ID NO: 357, LC CDR2 of SEQ ID NO: 366 and LCCDR3 of SEQ ID NO: 375 of CAR33-8; or

(ix) a LC CDR1 of SEQ ID NO: 358, LC CDR2 of SEQ ID NO: 367 and LC CDR3of SEQ ID NO: 376 of CAR33-9; and/or

(2) one, two, or three heavy chain (HC) CDRs chosen from one of thefollowing:

(i) a HC CDR1 of SEQ ID NO: 323, HC CDR2 of SEQ ID NO: 332 and HC CDR3of SEQ ID NO: 341 of CAR33-1;

(ii) a HC CDR1 of SEQ ID NO: 324, HC CDR2 of SEQ ID NO: 333 and HC CDR3of SEQ ID NO: 342 of CAR33-2;

(iii) a HC CDR1 of SEQ ID NO: 325, HC CDR2 of SEQ ID NO: 334 and HC CDR3of SEQ ID NO: 343 of CAR33-3;

(iv) a HC CDR1 of SEQ ID NO: 326, HC CDR2 of SEQ ID NO: 335 and HC CDR3of SEQ ID NO: 344 of CAR33-4;

(iv) a HC CDR1 of SEQ ID NO: 327, HC CDR2 of SEQ ID NO: 336 and HC CDR3of SEQ ID NO: 345 of CAR33-5;

(vi) a HC CDR1 of SEQ ID NO: 328, HC CDR2 of SEQ ID NO: 337 and HC CDR3of SEQ ID NO: 346 of CAR33-6;

(vii) a HC CDR1 of SEQ ID NO: 329, HC CDR2 of SEQ ID NO: 338 and HC CDR3of SEQ ID NO: 347 of CAR33-7;

(viii) a HC CDR1 of SEQ ID NO: 330, HC CDR2 of SEQ ID NO: 339 and HCCDR3 of SEQ ID NO: 348 of CAR33-8; or

(ix) a HC CDR1 of SEQ ID NO: 331, HC CDR2 of SEQ ID NO: 340 and HC CDR3of SEQ ID NO: 349 of CAR33-9.

In embodiments, fully human anti-CD33 single chain variable fragments(scFv) are generated and cloned into a lentiviral expression vector withthe intracellular CD3zeta chain and the intracellular co-stimulatorydomain of 4-1BB. Names of exemplary fully human anti-CD33 scFvs aredepicted in Table 1.

Construct ID CAR Nickname 141643 CD33-1 141644 CD33-2 141645 CD33-3141646 CD33-4 141647 CD33-5 141648 CD33-6 141649 CD33-7 141650 CD33-8141651 CD33-9

In embodiments, the order in which the VL and VH domains appear in thescFv is varied (i.e., VL-VH, or VH-VL orientation), and where eitherthree or four copies of the “G4S” (SEQ ID NO:25) subunit, in which eachsubunit comprises the sequence GGGGS (SEQ ID NO:25) (e.g., (G4S)₃ (SEQID NO:28) or (G4S)₄(SEQ ID NO:27)), connect the variable domains tocreate the entirety of the scFv domain, as shown in Table 3.

Exemplary sequences of the human scFv fragments (SEQ ID NOS: 39-83,including the optional leader sequence) are provided herein in Table 2.It is noted that the scFv fragments of SEQ ID NOs: 39-83, without aleader sequence (e.g., without the amino acid sequence of SEQ ID NO: 1or the nucleotide sequence of SEQ ID NO:12), are also encompassed by thepresent invention. Exemplary sequences of human scFv fragments, withoutthe leader sequence, are provided herein in Table 9 (SEQ ID NOS: 255-261for the nucleotide sequences, and SEQ ID NOS: 262-268 for the amino acidsequences).

leader (amino acid sequence) (SEQ ID NO: 1) MALPVTALLLPLALLLHAARPleader (nucleic acid sequence) (SEQ ID NO: 12)ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCT AGACCCCD8 hinge (amino acid sequence) (SEQ ID NO: 2)TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDCD8 hinge (nucleic acid sequence) (SEQ ID NO: 13)ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT CD8 transmembrane (amino acid sequence)(SEQ ID NO: 6) IYIWAPLAGTCGVLLLSLVITLYCCD8 transmembrane (nucleic acid sequence) (SEQ ID NO: 17)ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC 4-1BB Intracellular domain (amino acid sequence)(SEQ ID NO: 7) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL4-1BB Intracellular domain (nucleic acid sequence) (SEQ ID NO: 18)AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGG ATGTGAACTGCD28 Intracellular domain (amino acid sequence) (SEQ ID NO: 379)RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 379)CD28 Intracellular domain (nucleotide sequence) (SEQ ID NO: 380)AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTC (SEQ ID NO: 380)CCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC ICOS Intracellular domain (amino acid sequence)(SEQ ID NO: 381)T K K K Y S S S V H D P N G E Y M F M R A V N T A K K S R L T D V TL (SEQ ID NO: 381) ICOS Intracellular domain (nucleotide sequence)(SEQ ID NO: 382) ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTA (SEQ ID NO: 382)CD3 zeta domain (amino acid sequence) (SEQ ID NO: 9)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRCD3 zeta (nucleic acid sequence) (SEQ ID NO: 20)AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCD3 zeta domain (amino acid sequence; NCBI Reference Sequence NM_000734.3)(SEQ ID NO: 10) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRCD3 zeta (nucleic acid sequence; NCBI Reference Sequence NM_000734.3);(SEQ ID NO: 21) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC IgG4 Hinge (amino acid sequence)(SEQ ID NO: 36)ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMIgG4 Hinge (nucleotide sequence) (SEQ ID NO: 37)GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG

In embodiments, these clones contain a Q/K residue change in the signaldomain of the co-stimulatory domain derived from CD3zeta chain.

TABLE 2 Human CD33 CAR Constructs Name SEQ ID Sequence 141643 75ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCC CAR33-1CCAAGTGCAACTCGTCCAGTCCGGTGCAGAAGTCAAGAAGCCAGGAGAATCACTCAAGATTA Full - ntGCTGCAAAGGCAGCGGCTACTCCTTCACTTCCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGAAAGGGACTGGAGTGGATGGGAATCATCTACCCTGGCGATAGCGACACCAGATACTCCCCGAGCTTTCAAGGCCAAGTGACCATTTCGGCCGACAAGTCGATCTCCACCGCGTATCTGCAGTGGAGCTCACTGAAGGCTTCGGACACCGCCATGTACTACTGTGCCCGGCTGGGGGGAAGCCTGCCCGATTACGGAATGGACGTGTGGGGCCAGGGAACCATGGTCACTGTGTCCTCCGCCTCCGGGGGTGGAGGCTCCGGTGGAGGGGGGTCCGGTGGTGGAGGATCAGAAATTGTGCTGACCCAGTCTCCGCTGTCCTTGCCTGTGACCCCGGGCGAACCCGCAAGCATCTCCTGCCGGTCGTCGCAGTCCCTGCTTCACTCCAACGGCTACAACTACCTCGATTGGTACCTCCAGAAGCCTGGACAGAGCCCACAGCTGTTGATCTACCTGGGCTCGAACCGGGCCTCAGGAGTGCCGGACAGGTTCTCCGGCTCCGGGTCGGGAACCGACTTCACGCTGAAGATCTCCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTATTGCATGCAGGCGCTGCAGACCCTTATTACATTCGGACAGGGGACTAAGGTCGATATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 141643 48MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMP CAR33-1GKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLGGSL Full - aaPDYGMDVWGQGTMVTVSSASGGGGSGGGGSGGGGSEIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTLITFGQGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 141643 39MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMP CAR33-1GKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLGGSL scFv - aaPDYGMDVWGQGTMVTVSSASGGGGSGGGGSGGGGSEIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTLITFGQGTKVDIK 141643 57QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSP CAR33-1SFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLGGSLPDYGMDVWGQGTMVTVSS VH- aa141643 66 EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGCAR33-1 VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTLITFGQGTKVDIK VL - aa141644 76 ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCAR33-2 CCAAGTCCAACTCGTCCAATCAGGAGCTGAAGTCAAGAAGCCTGGAGCATCCGTGAGAGTGTFull - nt CCTGTAAAGCCTCCGGCTACATCTTCACCAACTACTACGTGCACTGGGTCAGACAGGCCCCGGGCCAGGGACTGGAATGGATGGGAATCATTTCCCCGTCCGGCGGATCGCCTACTTACGCGCAACGGCTGCAGGGCCGCGTGACCATGACTCGGGATCTCTCCACTTCAACCGTGTACATGGAACTGTCCAGCCTTACATCGGAGGATACTGCCGTGTACTTCTGCGCGAGGGAGTCCCGGCTGAGGGGCAACCGCCTCGGGCTGCAGTCAAGCATCTTCGATCACTGGGGCCAGGGCACCCTCGTGACCGTGTCCAGCGCCTCGGGGGGAGGAGGCTCCGGGGGCGGAGGTTCGGGCGGTGGTGGATCTGACATTCGCATGACTCAGTCCCCACCTTCACTGTCCGCTAGCGTGGGGGACCGCGTGACGATTCCGTGCCAAGCCAGCCAGGACATCAACAACCATCTGAACTGGTATCAGCAGAAGCCCGGAAAGGCCCCGCAGCTGCTGATCTACGACACCTCGAATCTGGAGATCGGCGTGCCATCCCGGTTCTCCGGTTCGGGAAGCGGAACCGACTTTACCCTGACTATCTCCTCCTTGCAACCCGAGGACATTGCCACCTACTACTGCCAGCAGTACGAAAACCTTCCCCTGACCTTCGGGGGTGGAACCAAAGTGGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 141644 49MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVRVSCKASGYIFTNYYVHWVRQAP CAR33-2GQGLEWMGIISPSGGSPTYAQRLQGRVTMTRDLSTSTVYMELSSLTSEDTAVYFCARESRLR Full - aaGNRLGLQSSIFDHWGQGTLVTVSSASGGGGSGGGGSGGGGSDIRMTQSPPSLSASVGDRVTIPCQASQDINNHLNWYQQKPGKAPQLLIYDTSNLEIGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQYENLPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 141644 40MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVRVSCKASGYIFTNYYVHWVRQAP CAR33-2GQGLEWMGIISPSGGSPTYAQRLQGRVTMTRDLSTSTVYMELSSLTSEDTAVYFCARESRLR scFv - aaGNRLGLQSSIFDHWGQGTLVTVSSASGGGGSGGGGSGGGGSDIRMTQSPPSLSASVGDRVTIPCQASQDINNHLNWYQQKPGKAPQLLIYDTSNLEIGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQYENLPLTFGGGTKVEIK 141644 58QVQLVQSGAEVKKPGASVRVSCKASGYIFTNYYVHWVRQAPGQGLEWMGIISPSGGSPTYAQ CAR33-2RLQGRVTMTRDLSTSTVYMELSSLTSEDTAVYFCARESRLRGNRLGLQSSIFDHWGQGTLVT VH - aaVSS 141644 67DIRMTQSPPSLSASVGDRVTIPCQASQDINNHLNWYQQKPGKAPQLLIYDTSNLEIGVPSRF CAR33-2SGSGSGTDFTLTISSLQPEDIATYYCQQYENLPLTFGGGTKVEIK VL - aa 141645 77ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCC CAR33-3CCAAGTGCAATTGGTGCAGTCAGGAGGAGGATTGGTGCAACCCGGAGGATCGCTGAGACTGT Full - ntCATGTGCTGCCAGCGGGTTCACATTCTCCTCCTACGCAATGTCCTGGGTCCGCCAGGCGCCGGGCAAAGGACTGGAATGGGTGTCCGCCATCTCGGGGTCGGGCGGCTCCACCTATTACGCTGACTCCGTGAAGGGACGCTTCACCATTAGCAGAGATAACTCCAAGAACACCCTCTACCTCCAAATGAACAGCCTTAGGGCTGAGGACACCGCCGTCTATTACTGCGCCAAGGAGGACACGATCCGGGGACCTAACTACTATTACTACGGAATGGATGTCTGGGGCCAGGGTACCACTGTGACCGTGTCCTCGGCCTCGGGAGGCGGAGGATCAGGGGGTGGTGGCTCTGGGGGGGGTGGCAGCGAAACTACTCTGACCCAGTCCCCCTCATCCGTGTCAGCGTCCGTGGGCGATCGGGTGTCGATCACTTGCCGGGCCTCCCAAGACATCGACACCTGGCTCGCGTGGTACCAGCTGAAGCCAGGAAAGGCCCCTAAGCTGCTGATGTACGCAGCCTCCAATCTGCAAGGAGGGGTGCCCTCCCGCTTTTCCGGGTCCGGCAGCGGAACCGACTTCATTCTGACTATCTCGAGCCTCCAGCCGGAGGATTTCGCCACCTACTACTGCCAGCAGGCCTCCATCTTCCCGCCGACTTTCGGTGGCGGAACCAAGGTCGACATTAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 141645 50MALPVTALLLPLALLLHAARPQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP CAR33-3GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEDTIR Full - aaGPNYYYYGMDVWGQGTTVTVSSASGGGGSGGGGSGGGGSETTLTQSPSSVSASVGDRVSITCRASQDIDTWLAWYQLKPGKAPKLLMYAASNLQGGVPSRFSGSGSGTDFILTISSLQPEDFATYYCQQASIFPPTFGGGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 141645 41MALPVTALLLPLALLLHAARPQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP CAR33-3GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEDTIR scFv- aaGPNYYYYGMDVWGQGTTVTVSSASGGGGSGGGGSGGGGSETTLTQSPSSVSASVGDRVSITCRASQDIDTWLAWYQLKPGKAPKLLMYAASNLQGGVPSRFSGSGSGTDFILTISSLQPEDFATYYCQQASIFPPTFGGGTKVDIK 141645 59QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYAD CAR33-3SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEDTIRGPNYYYYGMDVWGQGTTVTVS VH - aa S141645 68 ETTLTQSPSSVSASVGDRVSITCRASQDIDTWLAWYQLKPGKAPKLLMYAASNLQGGVPSRFCAR33-3 SGSGSGTDFILTISSLQPEDFATYYCQQASIFPPTFGGGTKVDIK VL - aa 141646 78ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCC CAR33-4CCAAGTGCAGCTCGTCCAATCCGGTGCAGAAGTGAAGAAGCCTGGCGAATCCCTGAAGATCT Full - ntCATGCAAAGGCTCGGGATACAGCTTCACCTCATATTGGATTGGATGGGTCAGACAGATGCCAGGAAAGGGTCTGGAGTGGATGGGAATCATCTACCCGGGAGACAGCGATACCCGGTACTCCCCGAGCTTCCAGGGACAGGTCACCATCTCGGCCGACAAGTCCATTACTACTGCCTACTTGCAATGGTCCTCGCTGCGCGCCTCCGATAGCGCCATGTACTACTGCGCGAGAGGCGGCTACTCCGACTACGACTACTACTTCGATTTCTGGGGACAGGGGACACTCGTGACTGTGTCCTCCGCGTCGGGTGGCGGCGGCTCGGGTGGAGGAGGAAGCGGAGGGGGAGGCTCCGAAATTGTGATGACCCAGTCACCCCTGTCGCTCCCTGTGACTCCTGGGGAACCGGCCTCCATCTCCTGCCGGAGCTCACAGAGCCTGCTGCACTCCAACGGATACAACTACCTCGATTGGTACCTTCAGAAGCCCGGCCAGTCGCCCCAGCTGCTGATCTACCTGGGGTCCAACCGGGCTAGCGGCGTGCCGGACCGCTTCTCCGGTTCCGGGTCTGGAACCGACTTCACGCTGAAAATCTCCAGGGTGGAGGCCGAGGACGTGGGAGTGTATTACTGTATGCAGGCCCTGCAAACCCCCTTCACCTTTGGCGGGGGCACCAAGGTCGAGATTAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 141646 51MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMP CAR33-4GKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSITTAYLQWSSLRASDSAMYYCARGGYSD Full - aaYDYYFDFWGQGTLVTVSSASGGGGSGGGGSGGGGSEIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPFTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 141646 42MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMP CAR33-4GKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSITTAYLQWSSLRASDSAMYYCARGGYSD scFv - aaYDYYFDFWGQGTLVTVSSASGGGGSGGGGSGGGGSEIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPFTFGGGTKVEIK 141646 60QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSP CAR33-4SFQGQVTISADKSITTAYLQWSSLRASDSAMYYCARGGYSDYDYYFDFWGQGTLVTVSS VH - aa141646 69 EIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGCAR33-4 VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPFTFGGGTKVEIK VL - aa141647 79 ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCAR33-5 CCAAGTGCAACTCGTCCAAAGCGGTGGAGATCTCGCCCAGCCCGGAAGATCCCTTAGACTCTFull - nt CATGTGCCGCCAGCGGGTTCACCTTCGACGACTACGCTATGCATTGGGTGCGCCAGGCCCCGGGGAAGGGACTGGAATGGGTGGCCGTGATTTGGCCGGACGGCGGACAGAAGTACTACGGAGACAGCGTGAAAGGGCGGTTCACCGTGTCGAGGGACAACCCGAAGAATACCCTCTACCTTCAAATGAACTCCCTGCGCGCCGAGGACACCGCGATCTACTACTGCGTGCGCCACTTTAACGCATGGGATTACTGGGGACAGGGGACTCTGGTCACTGTGTCCTCCGCCTCTGGCGGCGGAGGTTCCGGCGGTGGTGGCTCCGGTGGAGGAGGATCGGACATCCAGCTGACCCAGTCCCCTTCCTCACTGTCGGCGTACGTGGGAGGCCGGGTCACTATCACGTGCCAGGCATCCCAGGGCATTTCCCAGTTCCTGAACTGGTTCCAGCAGAAGCCCGGAAAGGCCCCTAAGCTGTTGATTTCCGATGCTAGCAACCTGGAACCCGGCGTGCCGTCACGGTTCAGCGGCTCCGGGTCGGGCACCGACTTCACCTTCACCATCACTAACCTCCAACCGGAGGACATCGCCACCTATTACTGCCAGCAGTACGATGATCTGCCACTGACTTTCGGCGGCGGAACCAAGGTCGAAATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 141647 52MALPVTALLLPLALLLHAARPQVQLVQSGGDLAQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVAVIWPDGGQKYYGDSVKGRFTVSRDNPKNTLYLQMNSLRAEDTAIYYCVRHFNAW CAR33-5DYWGQGTLVTVSSASGGGGSGGGGSGGGGSDIQLTQSPSSLSAYVGGRVTITCQASQGISQF Full - aaLNWFQQKPGKAPKLLISDASNLEPGVPSRFSGSGSGTDFTFTITNLQPEDIATYYCQQYDDLPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 141647 43MALPVTALLLPLALLLHAARPQVQLVQSGGDLAQPGRSLRLSCAASGFTFDDYAMHWVRQAP CAR33-5GLEWVAVIWPDGGQKYYGDSVKGRFTVSRDNPKNTLYLQMNSLRAEDTAIYYCVRHFNAW scFv- aaDYWGQGTLVTVSSASGGGGSGGGGSGGGGSDIQLTQSPSSLSAYVGGRVTITCQASQGISQFLNWFQQKPGKAPKLLISDASNLEPGVPSRFSGSGSGTDFTFTITNLQPEDIATYYCQQYDDLPLTFGGGTKVEIK 141647 61QVQLVQSGGDLAQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVAVIWPDGGQKYYGD CAR33-5SVKGRFTVSRDNPKNTLYLQMNSLRAEDTAIYYCVRHFNAWDYWGQGTLVTVSS VH - aa 141647 70DIQLTQSPSSLSAYVGGRVTITCQASQGISQFLNWFQQKPGKAPKLLISDASNLEPGVPSRF CAR33-5SGSGSGTDFTFTITNLQPEDIATYYCQQYDDLPLTFGGGTKVEIK VL - aa 141648 80ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCC CAR33-6CCAAGTGCAACTCGTCCAATCCGGTGGTGGTGTCGTGCAACCAGGAAAGTCTCTTCGCCTCT Full - ntCATGCGCTGCCAGCGGATTCACGTTTTCCATCTTCGCTATGCACTGGGTGCGGCAGGCCCCGGGAAAGGGACTGGAATGGGTGGCAACCATTTCATACGATGGATCAAACGCGTTCTACGCCGACTCCGTGGAAGGAAGGTTCACCATCTCGAGAGACAACTCCAAGGACTCGCTGTATCTGCAAATGGACTCCCTGCGCCCTGAGGATACCGCCGTCTACTACTGCGTGAAGGCCGGCGACGGGGGATACGACGTGTTCGATTCGTGGGGCCAGGGAACTCTGGTCACCGTGTCCAGCGCGAGCGGGGGAGGCGGATCGGGTGGTGGAGGGTCCGGGGGAGGAGGCTCCGAGATCGTGATGACTCAGTCGCCGCTCTCCCTCCCCGTGACCCCCGGAGAGCCAGCTAGCATTTCATGTCGGAGCTCCCAGTCCCTGCTGCACTCCAACGGCTACAATTACCTGGATTGGTACTTGCAGAAGCCTGGGCAGAGCCCTCAGCTGCTGATCTACCTCGGCTCGAACAGAGCCTCCGGCGTGCCGGACCGGTTTTCCGGGAGCGGCAGCGGCACCGATTTCACCTTGAAAATCTCCCGCGTGGAAGCCGAGGACGTGGGCGTGTACTATTGCATGCAGGCCCTGCAGACTCCCACCTTCGGCCCGGGAACTAAGGTCGACATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 141648 53MALPVTALLLPLALLLHAARPQVQLVQSGGGVVQPGKSLRLSCAASGFTFSIFAMHWVRQAP CAR33-6GKGLEWVATISYDGSNAFYADSVEGRFTISRDNSKDSLYLQMDSLRPEDTAVYYCVKAGDGG Full - aaYDVFDSWGQGTLVTVSSASGGGGSGGGGSGGGGSEIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPTFGPGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 141648 44MALPVTALLLPLALLLHAARPQVQLVQSGGGVVQPGKSLRLSCAASGFTFSIFAMHWVRQAP CAR33-6GKGLEWVATISYDGSNAFYADSVEGRFTISRDNSKDSLYLQMDSLRPEDTAVYYCVKAGDGG scFv - aaYDVFDSWGQGTLVTVSSASGGGGSGGGGSGGGGSEIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPTFGPGTKVDIK 141648 62QVQLVQSGGGVVQPGKSLRLSCAASGFTFSIFAMHWVRQAPGKGLEWVATISYDGSNAFYAD CAR33-6SVEGRFTISRDNSKDSLYLQMDSLRPEDTAVYYCVKAGDGGYDVFDSWGQGTLVTVSS VH - aa141648 71 EIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGCAR33-6 VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPTFGPGTKVDIK VL - aa 14164981 ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCAR33-7 CGAAGTGCAATTGGTGGAATCTGGAGGAGGATTGGTGCAACCTGGAGGATCTCTGAGACTGTFull - nt CATGTGCCGCCAGCGGCTTCACATTTTCCTCCTACGCGATGTCATGGGTCCGCCAGGCACCGGGGAAAGGACTGGAATGGGTGTCCGCCATTTCGGGATCGGGAGGCTCGACCTACTACGCCGACAGCGTGAAGGGAAGATTCACTATCTCCCGGGATAACTCCAAGAATACTCTGTATCTCCAAATGAACTCCCTGAGGGCCGAGGATACTGCCGTGTACTACTGCGCTAAGGAAACCGACTACTACGGCTCAGGAACCTTCGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCGGCCTCCGGCGGCGGAGGTTCGGGGGGGGGCGGTTCCGGGGGAGGGGGCAGCGACATCCAGATGACCCAGTCCCCAAGCTCCCTTTCCGCGTCCGTGGGAGATCGCGTGACCATTTCGTGCCGGGCTAGCCAGGGCATCGGTATCTATCTTGCGTGGTACCAGCAGCGGAGCGGAAAGCCGCCCCAGCTGCTGATCCACGGCGCCTCAACTCTGCAATCCGGGGTCCCCAGCCGGTTCAGCGGTAGCGGGTCGGGTACCGACTTTACCCTGACTATCTCCTCCCTCCAACCGGAGGACTTCGCCTCCTACTGGTGCCAGCAGTCCAACAACTTCCCTCCCACCTTCGGCCAGGGAACGAAGGTCGAGATTAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 141649 54MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP CAR33-7GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKETDYY Full - aaGSGTFDYWGQGTLVTVSSASGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTISCRASQGIGIYLAWYQQRSGKPPQLLIHGASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFASYWCQQSNNFPPTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 141649 45MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP CAR33-7GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKETDYY scFv - aaGSGTFDYWGQGTLVTVSSASGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTISCRASQGIGIYLAWYQQRSGKPPQLLIHGASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFASYWCQQSNNFPPTFGQGTKVEIK 141649 63EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYAD CAR33-7SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKETDYYGSGTFDYWGQGTLVTVSS VH - aa141649 72 DIQMTQSPSSLSASVGDRVTISCRASQGIGIYLAWYQQRSGKPPQLLIHGASTLQSGVPSRFCAR33-7 SGSGSGTDFTLTISSLQPEDFASYWCQQSNNFPPTFGQGTKVEIK VL - aa 141650 82ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCC CAR33-8CCAAGTCCAACTCGTCCAGTCCGGTGCAGAAGTCAAGAAGCCAGGAGCCTCCGTGAGAGTGT Full - ntCGTGCAAAGCGTCCGGCTACATGTTCACCGACTTTTTCATTCACTGGGTGCGCCAGGCGCCCGGACAGGGTCTGGAGTGGATGGGGTGGATCAACCCTAACTCCGGCGTGACTAAATACGCCCAGAAGTTCCAGGGCCGCGTGACCATGACCCGGAACACTAGCATCTCCACCGCCTACATGGAACTGTCATCCCTCCGGTCCGAGGATACCGCCGTGTACTACTGCGCCACCTGGTACAGCAGCGGTTGGTACGGCATCGCGAACATTTGGGGACAGGGGACTATGGTCACCGTGTCATCCGCCTCCGGGGGAGGAGGGTCCGGCGGCGGAGGTTCTGGAGGAGGCGGCTCGGACATCCAGTTGACGCAGAGCCCCTCGTCGCTGAGCGCCTCCGTGGGCGACAGAGTGACCATTACCTGTCAAGCTTCCCATGATATCTCGAACTACCTCCACTGGTATCAGCAGAAGCCGGGAAAGGCTCCCAAGCTGCTGATCTACGACGCCTCCAATCTGGAAACCGGAGTGCCGAGCCGGTTCACTGGATCAGGGAGCGGCACTGACTTCACCCTGACAATTAGGTCGCTGCAGCCGGAGGATGTGGCAGCCTACTACTGCCAACAGTCAGACGACCTTCCTCACACTTTCGGACAAGGGACTAAGGTCGACATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 141650 55MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVRVSCKASGYMFTDFFIHWVRQAP CAR33-8GQGLEWMGWINPNSGVTKYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCATWYSSG Full - aaWYGIANIWGQGTMVTVSSASGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCQASHDISNYLHWYQQKPGKAPKLLIYDASNLETGVPSRFTGSGSGTDFTLTIRSLQPEDVAAYYCQQSDDLPHTFGQGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 141650 46MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVRVSCKASGYMFTDFFIHWVRQAP CAR33-8GQGLEWMGWINPNSGVTKYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCATWYSSG scFv - aaWYGIANIWGQGTMVTVSSASGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCQASHDISNYLHWYQQKPGKAPKLLIYDASNLETGVPSRFTGSGSGTDFTLTIRSLQPEDVAAYYCQQSDDLPHTFGQGTKVDIK 141650 64QVQLVQSGAEVKKPGASVRVSCKASGYMFTDFFIHWVRQAPGQGLEWMGWINPNSGVTKYAQ CAR33-8KFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCATWYSSGWYGIANIWGQGTMVTVSS VH - aa141650 73 DIQLTQSPSSLSASVGDRVTITCQASHDISNYLHWYQQKPGKAPKLLIYDASNLETGVPSRFCAR33-8 TGSGSGTDFTLTIRSLQPEDVAAYYCQQSDDLPHTFGQGTKVDIK VL - aa 141651 83ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCC CAR33-9CCAAGTGCAACTCGTCCAGTCCGGTGCAGAAGTGAAAAAGCCAGGAGAAAGCCTCAAGATCA Full - ntGCTGCAAGGGATCTGGGTACAGCTTCACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGAAAGGGACTGGAGTGGATGGGCATTATCTACCCTGGGGACTCCGACACCCGGTATTCCCCGAGCTTCCAAGGACAGGTCACCATCTCCGCCGATAAGTCGATTAGCACTGCGTACTTGCAGTGGTCAAGCCTGAAGGCCTCGGACACCGCCATGTACTACTGCGCGAGACACGGGCCCTCGTCCTGGGGCGAATTTGACTACTGGGGCCAGGGAACGCTTGTGACCGTGTCGTCCGCGTCCGGGGGTGGAGGATCAGGAGGAGGAGGCTCCGGTGGTGGCGGTAGCGACATCCGGCTGACTCAGTCCCCTTCCTCACTCTCCGCCTCCGTGGGGGACCGCGTGACCATTACCTGTCGGGCATCACAGTCCATCAGCTCATACCTGAACTGGTATCAGCAGAAGCCGGGGAAGGCCCCGAAACTCCTGATCTACGCCGCCTCCTCCCTGCAATCCGGCGTGCCCTCGAGGTTCTCCGGCTCCGGCTCGGGAACCGATTTCACTCTGACAATTAGCAGCCTGCAGCCTGAGGATTTCGCTACCTACTACTGCCAGCAGTCCTACTCGACTCCGCTGACTTTCGGCGGGGGAACCAAGGTCGACATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 141651 56MALPVTALLLPLALLLHAARPQVQLVQ SGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMP CAR33-9GKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGPSSWGEFDYWGQGTLVTVSSASGGGGSGGGGSGGGGSDIRLTQSPSSLSASVGDRVTITCRASQS Full - aaISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 141651 47MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMP CAR33-9GKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGPSS scFv - aaWGEFDYWGQGTLVTVSSASGGGGSGGGGSGGGGSDIRLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIK 141651 65QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSP CAR33-9SFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGPSSWGEFDYWGQGTLVTVSS VH - aa141651 74 DIRLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFCAR33-9 SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIK VL - aa

TABLE 9 Human CD33 CAR scFv sequences Name SEQ ID Sequence 141643 255CAAGTGCAACTCGTCCAGTCCGGTGCAGAAGTCAAGAAGCCAGGAGAATCACTCAAGATTAGCT(CD33-1)GCAAAGGCAGCGGCTACTCCTTCACTTCCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGAAAscFv - ntGGGACTGGAGTGGATGGGAATCATCTACCCTGGCGATAGCGACACCAGATACTCCCCGAGCTTTCAAGGCCAAGTGACCATTTCGGCCGACAAGTCGATCTCCACCGCGTATCTGCAGTGGAGCTCACTGAAGGCTTCGGACACCGCCATGTACTACTGTGCCCGGCTGGGGGGAAGCCTGCCCGATTACGGAATGGACGTGTGGGGCCAGGGAACCATGGTCACTGTGTCCTCCGCCTCCGGGGGTGGAGGCTCCGGTGGAGGGGGGTCCGGTGGTGGAGGATCAGAAATTGTGCTGACCCAGTCTCCGCTGTCCTTGCCTGTGACCCCGGGCGAACCCGCAAGCATCTCCTGCCGGTCGTCGCAGTCCCTGCTTCACTCCAACGGCTACAACTACCTCGATTGGTACCTCCAGAAGCCTGGACAGAGCCCACAGCTGTTGATCTACCTGGGCTCGAACCGGGCCTCAGGAGTGCCGGACAGGTTCTCCGGCTCCGGGTCGGGAACCGACTTCACGCTGAAGATCTCCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTATTGCATGCAGGCGCTGCAGACCCTTATTACATTCGGACAGGGGACTAAGGTCGATATCAAG 141643 262QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSF(CD33-1)QGQVTISADKSISTAYLQWSSLKASDTAMYYCARLGGSLPDYGMDVWGQGTMVTVSSASGGGGSscFv - aaGGGGSGGGGSEIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTLITFGQGTKVDIK 141644 256CAAGTCCAACTCGTCCAATCAGGAGCTGAAGTCAAGAAGCCTGGAGCATCCGTGAGAGTGTCCT(CD33-2)GTAAAGCCTCCGGCTACATCTTCACCAACTACTACGTGCACTGGGTCAGACAGGCCCCGGGCCAscFv - ntGGGACTGGAATGGATGGGAATCATTTCCCCGTCCGGCGGATCGCCTACTTACGCGCAACGGCTGCAGGGCCGCGTGACCATGACTCGGGATCTCTCCACTTCAACCGTGTACATGGAACTGTCCAGCCTTACATCGGAGGATACTGCCGTGTACTTCTGCGCGAGGGAGTCCCGGCTGAGGGGCAACCGCCTCGGGCTGCAGTCAAGCATCTTCGATCACTGGGGCCAGGGCACCCTCGTGACCGTGTCCAGCGCCTCGGGGGGAGGAGGCTCCGGGGGCGGAGGTTCGGGCGGTGGTGGATCTGACATTCGCATGACTCAGTCCCCACCTTCACTGTCCGCTAGCGTGGGGGACCGCGTGACGATTCCGTGCCAAGCCAGCCAGGACATCAACAACCATCTGAACTGGTATCAGCAGAAGCCCGGAAAGGCCCCGCAGCTGCTGATCTACGACACCTCGAATCTGGAGATCGGCGTGCCATCCCGGTTCTCCGGTTCGGGAAGCGGAACCGACTTTACCCTGACTATCTCCTCCTTGCAACCCGAGGACATTGCCACCTACTACTGCCAGCAGTACGAAAACCTTCCCCTGACCTTCGGGGGTGGAACCAAAGTGGAGATCAAG 141644 263QVQLVQSGAEVKKPGASVRVSCKASGYIFTNYYVHWVRQAPGQGLEWMGIISPSGGSPTYAQRL(CD33-2)QGRVTMTRDLSTSTVYMELSSLTSEDTAVYFCARESRLRGNRLGLQSSIFDHWGQGTLVTVSSAscFv - aaSGGGGSGGGGSGGGGSDIRMTQSPPSLSASVGDRVTIPCQASQDINNHLNWYQQKPGKAPQLLIYDTSNLEIGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQYENLPLTFGGGTKVEIK 141646 257CAAGTGCAGCTCGTCCAATCCGGTGCAGAAGTGAAGAAGCCTGGCGAATCCCTGAAGATCTCAT(CD33-4)GCAAAGGCTCGGGATACAGCTTCACCTCATATTGGATTGGATGGGTCAGACAGATGCCAGGAAAscFv - ntGGGTCTGGAGTGGATGGGAATCATCTACCCGGGAGACAGCGATACCCGGTACTCCCCGAGCTTCCAGGGACAGGTCACCATCTCGGCCGACAAGTCCATTACTACTGCCTACTTGCAATGGTCCTCGCTGCGCGCCTCCGATAGCGCCATGTACTACTGCGCGAGAGGCGGCTACTCCGACTACGACTACTACTTCGATTTCTGGGGACAGGGGACACTCGTGACTGTGTCCTCCGCGTCGGGTGGCGGCGGCTCGGGTGGAGGAGGAAGCGGAGGGGGAGGCTCCGAAATTGTGATGACCCAGTCACCCCTGTCGCTCCCTGTGACTCCTGGGGAACCGGCCTCCATCTCCTGCCGGAGCTCACAGAGCCTGCTGCACTCCAACGGATACAACTACCTCGATTGGTACCTTCAGAAGCCCGGCCAGTCGCCCCAGCTGCTGATCTACCTGGGGTCCAACCGGGCTAGCGGCGTGCCGGACCGCTTCTCCGGTTCCGGGTCTGGAACCGACTTCACGCTGAAAATCTCCAGGGTGGAGGCCGAGGACGTGGGAGTGTATTACTGTATGCAGGCCCTGCAAACCCCCTTCACCTTTGGCGGGGGCACCAAGGTCGAGATTAAG 141646 264QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSF(CD33-4)QGQVTISADKSITTAYLQWSSLRASDSAMYYCARGGYSDYDYYFDFWGQGTLVTVSSASGGGGSscFv - aaGGGGSGGGGSEIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPFTFGGGTKVEIK 141647 258CAAGTGCAACTCGTCCAAAGCGGTGGAGATCTCGCCCAGCCCGGAAGATCCCTTAGACTCTCAT(CD33-5)GTGCCGCCAGCGGGTTCACCTTCGACGACTACGCTATGCATTGGGTGCGCCAGGCCCCGGGGAAscFv - ntGGGACTGGAATGGGTGGCCGTGATTTGGCCGGACGGCGGACAGAAGTACTACGGAGACAGCGTGAAAGGGCGGTTCACCGTGTCGAGGGACAACCCGAAGAATACCCTCTACCTTCAAATGAACTCCCTGCGCGCCGAGGACACCGCGATCTACTACTGCGTGCGCCACTTTAACGCATGGGATTACTGGGGACAGGGGACTCTGGTCACTGTGTCCTCCGCCTCTGGCGGCGGAGGTTCCGGCGGTGGTGGCTCCGGTGGAGGAGGATCGGACATCCAGCTGACCCAGTCCCCTTCCTCACTGTCGGCGTACGTGGGAGGCCGGGTCACTATCACGTGCCAGGCATCCCAGGGCATTTCCCAGTTCCTGAACTGGTTCCAGCAGAAGCCCGGAAAGGCCCCTAAGCTGTTGATTTCCGATGCTAGCAACCTGGAACCCGGCGTGCCGTCACGGTTCAGCGGCTCCGGGTCGGGCACCGACTTCACCTTCACCATCACTAACCTCCAACCGGAGGACATCGCCACCTATTACTGCCAGCAGTACGATGATCTGCCACTGACTTTCGGCGGCGGAACCAAGGTCGAAATCAAG 141647 265QVQLVQSGGDLAQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVAVIWPDGGQKYYGDSV (CD335)KGRFTVSRDNPKNTLYLQMNSLRAEDTAIYYCVRHFNAWDYWGQGTLVTVSSASGGGGSGGGGSscFv - aaGGGGSDIQLTQSPSSLSAYVGGRVTITCQASQGISQFLNWFQQKPGKAPKLLISDASNLEPGVPSRFSGSGSGTDFTFTITNLQPEDIATYYCQQYDDLPLTFGGGTKVEIK 141648 259CAAGTGCAACTCGTCCAATCCGGTGGTGGTGTCGTGCAACCAGGAAAGTCTCTTCGCCTCTCAT(CD33-6)GCGCTGCCAGCGGATTCACGTTTTCCATCTTCGCTATGCACTGGGTGCGGCAGGCCCCGGGAAAscFv - ntGGGACTGGAATGGGTGGCAACCATTTCATACGATGGATCAAACGCGTTCTACGCCGACTCCGTGGAAGGAAGGTTCACCATCTCGAGAGACAACTCCAAGGACTCGCTGTATCTGCAAATGGACTCCCTGCGCCCTGAGGATACCGCCGTCTACTACTGCGTGAAGGCCGGCGACGGGGGATACGACGTGTTCGATTCGTGGGGCCAGGGAACTCTGGTCACCGTGTCCAGCGCGAGCGGGGGAGGCGGATCGGGTGGTGGAGGGTCCGGGGGAGGAGGCTCCGAGATCGTGATGACTCAGTCGCCGCTCTCCCTCCCCGTGACCCCCGGAGAGCCAGCTAGCATTTCATGTCGGAGCTCCCAGTCCCTGCTGCACTCCAACGGCTACAATTACCTGGATTGGTACTTGCAGAAGCCTGGGCAGAGCCCTCAGCTGCTGATCTACCTCGGCTCGAACAGAGCCTCCGGCGTGCCGGACCGGTTTTCCGGGAGCGGCAGCGGCACCGATTTCACCTTGAAAATCTCCCGCGTGGAAGCCGAGGACGTGGGCGTGTACTATTGCATGCAGGCCCTGCAGACTCCCACCTTCGGCCCGGGAACTAAGGTCGACATCAAG 141648 266QVQLVQSGGGVVQPGKSLRLSCAASGFTFSIFAMHWVRQAPGKGLEWVATISYDGSNAFYADSV(CD33-6)EGRFTISRDNSKDSLYLQMDSLRPEDTAVYYCVKAGDGGYDVFDSWGQGTLVTVSSASGGGGSGscFv - aaGGGSGGGGSEIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPTFGPGTKVDIK 141649 260GAAGTGCAATTGGTGGAATCTGGAGGAGGATTGGTGCAACCTGGAGGATCTCTGAGACTGTCAT(CD33-7)GTGCCGCCAGCGGCTTCACATTTTCCTCCTACGCGATGTCATGGGTCCGCCAGGCACCGGGGAAscFv - ntAGGACTGGAATGGGTGTCCGCCATTTCGGGATCGGGAGGCTCGACCTACTACGCCGACAGCGTGAAGGGAAGATTCACTATCTCCCGGGATAACTCCAAGAATACTCTGTATCTCCAAATGAACTCCCTGAGGGCCGAGGATACTGCCGTGTACTACTGCGCTAAGGAAACCGACTACTACGGCTCAGGAACCTTCGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCGGCCTCCGGCGGCGGAGGTTCGGGGGGGGGCGGTTCCGGGGGAGGGGGCAGCGACATCCAGATGACCCAGTCCCCAAGCTCCCTTTCCGCGTCCGTGGGAGATCGCGTGACCATTTCGTGCCGGGCTAGCCAGGGCATCGGTATCTATCTTGCGTGGTACCAGCAGCGGAGCGGAAAGCCGCCCCAGCTGCTGATCCACGGCGCCTCAACTCTGCAATCCGGGGTCCCCAGCCGGTTCAGCGGTAGCGGGTCGGGTACCGACTTTACCCTGACTATCTCCTCCCTCCAACCGGAGGACTTCGCCTCCTACTGGTGCCAGCAGTCCAACAACTTCCCTCCCACCTTCGGCCAGGGAACGAAGGTCGAGATTAAG 141649 267EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSV(CD33-7)KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKETDYYGSGTFDYWGQGTLVTVSSASGGGGSscFv - aaGGGGSGGGGSDIQMTQSPSSLSASVGDRVTISCRASQGIGIYLAWYQQRSGKPPQLLIHGASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFASYWCQQSNNFPPTFGQGTKVEIK 141651 261CAAGTGCAACTCGTCCAGTCCGGTGCAGAAGTGAAAAAGCCAGGAGAAAGCCTCAAGATCAGCT(CD33-9)GCAAGGGATCTGGGTACAGCTTCACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGAAAscFv - ntGGGACTGGAGTGGATGGGCATTATCTACCCTGGGGACTCCGACACCCGGTATTCCCCGAGCTTCCAAGGACAGGTCACCATCTCCGCCGATAAGTCGATTAGCACTGCGTACTTGCAGTGGTCAAGCCTGAAGGCCTCGGACACCGCCATGTACTACTGCGCGAGACACGGGCCCTCGTCCTGGGGCGAATTTGACTACTGGGGCCAGGGAACGCTTGTGACCGTGTCGTCCGCGTCCGGGGGTGGAGGATCAGGAGGAGGAGGCTCCGGTGGTGGCGGTAGCGACATCCGGCTGACTCAGTCCCCTTCCTCACTCTCCGCCTCCGTGGGGGACCGCGTGACCATTACCTGTCGGGCATCACAGTCCATCAGCTCATACCTGAACTGGTATCAGCAGAAGCCGGGGAAGGCCCCGAAACTCCTGATCTACGCCGCCTCCTCCCTGCAATCCGGCGTGCCCTCGAGGTTCTCCGGCTCCGGCTCGGGAACCGATTTCACTCTGACAATTAGCAGCCTGCAGCCTGAGGATTTCGCTACCTACTACTGCCAGCAGTCCTACTCGACTCCGCTGACTTTCGGCGGGGGAACCAAGGTCGACATCAAG 141651 268QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGITYPGDSDTRYSPSF(CD33-9)QGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGPSSWGEFDYWGQGTLVTVSSASGGGGSGscFv - aaGGGSGGGGSDIRLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVDIK

In embodiments, CAR scFv fragments are cloned into lentiviral vectors tocreate a full length CAR construct in a single coding frame, and using apromoter, e.g., EF1 alpha promoter, for expression (SEQ ID NO: 11).

SEQ ID NO: 11 EF1 alpha promoterCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA Gly/Ser (SEQ ID NO: 25) GGGGSGly/Ser (SEQ ID NO: 26):This sequence may encompass 1-6 “Gly Gly Gly Gly Ser” repeating unitsGGGGSGGGGS GGGGSGGGGS GGGGSGGGGS Gly/Ser (SEQ ID NO: 27)GGGGSGGGGS GGGGSGGGGS Gly/Ser (SEQ ID NO: 28) GGGGSGGGGS GGGGSGly/Ser (SEQ ID NO: 29) GGGS PolyA: (A)₅₀₀₀ (SEQ ID NO: 30)This sequence may encompass 50-5000 adenines.PolyA: (T)₁₀₀ (SEQ ID NO: 31) PolyA: (T)₅₀₀₀ (SEQ ID NO: 32)This sequence may encompass 50-5000 thymines.PolyA: (A)₅₀₀₀ (SEQ ID NO: 33)This sequence may encompass 100-5000 adenines.PolyA: (A)₄₀₀ (SEQ ID NO: 34) PolyA: (A)₂₀₀₀ (SEQ ID NO: 35)Gly/Ser (SEQ ID NO: 38):This sequence may encompass 1-10 “Gly Gly Gly Ser” repeating unitsGGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS

Additional examples of CAR molecules or antibody fragments thereof areprovided in Example 3. Murine and humanized versions of an anti-CD33antibody, 2213, are disclosed. For example, Example 3 provides thefollowing: the nucleotide sequence of 2213 murine anti-CD33 IgG4nucleotide sequence (SEQ ID NO: 138); the 2213 CAR nucleotide sequence(SEQ ID NO: 139); the 2213 CAR amino acid sequence (SEQ ID NO: 140); the2213 scFv nucleotide sequence (SEQ ID NO: 141); and the 2213 scFv aminoacid sequence (SEQ ID NO: 142); the 2218 humanized anti-CD33 IgG4Hnucleotide sequence (SEQ ID NO: 143).

Other embodiments disclosed in Example 3 include CAR molecules andanti-CD33 antibody fragments of Gemtuzumab ozogamicin previouslymarketed as Mylotarg) (e.g., the humanized version described herein as“humanized my96”). The amino acid sequence of anti-CD33 scFv ofGemtuzumab ozogamicin (an immunoconjugate targeting CD33) with 41BB andCD3 zeta signaling domains is described in Example 3; and SEQ ID NO:145. The corresponding nucleotide sequence of humanized my96 is depictedas SEQ ID NO: 144. The humanized my96 nucleotide sequence is providedherein as SEQ ID NO: 146, and the amino acid sequence is SEQ ID NO: 147.

In one embodiment, the CD33 CAR and CD33 CART described herein comprisean antigen binding domain comprising one or more, e.g., one, two, orthree, CDRs of the heavy chain variable domain and/or one or more, e.g.,one, two, or three, CDRs of the light chain variable domain, or the VHor VL of the scFv sequence encoded by GenBank reference no. AM402974.1(See, Wang et al., Mol. Ther., vol. 23:1, pp. 184-191 (2015), herebyincorporated by reference.

The CAR scFv fragments can be cloned into lentiviral vectors to create afull length CAR construct in a single coding frame, and using the EF1alpha promoter for expression (SEQ ID NO: 11).

The CAR construct can include a Gly/Ser linker having one or more of thefollowing sequences: GGGGS (SEQ ID NO:25); encompassing 1-6 “Gly Gly GlyGly Ser” repeating units, e.g., GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS (SEQ IDNO:26); GGGGSGGGGS GGGGSGGGGS (SEQ ID NO:27); GGGGSGGGGS GGGGS (SEQ IDNO:28); GGGS (SEQ ID NO:29); or encompassing 1-10 “Gly Gly Gly Ser”repeating units, e.g., GGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS (SEQID NO:38). In embodiments, the CAR construct include a poly A sequence,e.g., a sequence encompassing 50-5000 or 100-5000 adenines (e.g., SEQ IDNO:30, SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:35), or a sequenceencompassing 50-5000 thymines (e.g., SEQ ID NO:31, SEQ ID NO:32).Alternatively, the CAR construct can include, for example, a linkerincluding the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 383)

Bispecific CARs

In an embodiment a multispecific antibody molecule is a bispecificantibody molecule. A bispecific antibody has specificity for no morethan two antigens. A bispecific antibody molecule is characterized by afirst immunoglobulin variable domain sequence which has bindingspecificity for a first epitope and a second immunoglobulin variabledomain sequence that has binding specificity for a second epitope. In anembodiment the first and second epitopes are on the same antigen, e.g.,the same protein (or subunit of a multimeric protein). In an embodimentthe first and second epitopes overlap. In an embodiment the first andsecond epitopes do not overlap. In an embodiment the first and secondepitopes are on different antigens, e.g., different proteins (ordifferent subunits of a multimeric protein). In an embodiment abispecific antibody molecule comprises a heavy chain variable domainsequence and a light chain variable domain sequence which have bindingspecificity for a first epitope and a heavy chain variable domainsequence and a light chain variable domain sequence which have bindingspecificity for a second epitope. In an embodiment a bispecific antibodymolecule comprises a half antibody having binding specificity for afirst epitope and a half antibody having binding specificity for asecond epitope. In an embodiment a bispecific antibody moleculecomprises a half antibody, or fragment thereof, having bindingspecificity for a first epitope and a half antibody, or fragmentthereof, having binding specificity for a second epitope. In anembodiment a bispecific antibody molecule comprises a scFv, or fragmentthereof, have binding specificity for a first epitope and a scFv, orfragment thereof, have binding specificity for a second epitope.

In certain embodiments, the antibody molecule is a multi-specific (e.g.,a bispecific or a trispecific) antibody molecule. Protocols forgenerating bispecific or heterodimeric antibody molecules are known inthe art; including but not limited to, for example, the “knob in a hole”approach described in, e.g., U.S. Pat. No. 5,731,168; the electrostaticsteering Fc pairing as described in, e.g., WO 09/089004, WO 06/106905and WO 2010/129304; Strand Exchange Engineered Domains (SEED)heterodimer formation as described in, e.g., WO 07/110205; Fab armexchange as described in, e.g., WO 08/119353, WO 2011/131746, and WO2013/060867; double antibody conjugate, e.g., by antibody cross-linkingto generate a bi-specific structure using a heterobifunctional reagenthaving an amine-reactive group and a sulfhydryl reactive group asdescribed in, e.g., U.S. Pat. No. 4,433,059; bispecific antibodydeterminants generated by recombining half antibodies (heavy-light chainpairs or Fabs) from different antibodies through cycle of reduction andoxidation of disulfide bonds between the two heavy chains, as describedin, e.g., U.S. Pat. No. 4,444,878; trifunctional antibodies, e.g., threeFab fragments cross-linked through sulfhydryl reactive groups, asdescribed in, e.g., U.S. Pat. No. 5,273,743; biosynthetic bindingproteins, e.g., pair of scFvs cross-linked through C-terminal tailspreferably through disulfide or amine-reactive chemical cross-linking,as described in, e.g., U.S. Pat. No. 5,534,254; bifunctional antibodies,e.g., Fab fragments with different binding specificities dimerizedthrough leucine zippers (e.g., c-fos and c-jun) that have replaced theconstant domain, as described in, e.g., U.S. Pat. No. 5,582,996;bispecific and oligospecific mono- and oligovalent receptors, e.g.,VH-CH1 regions of two antibodies (two Fab fragments) linked through apolypeptide spacer between the CH1 region of one antibody and the VHregion of the other antibody typically with associated light chains, asdescribed in, e.g., U.S. Pat. No. 5,591,828; bispecific DNA-antibodyconjugates, e.g., crosslinking of antibodies or Fab fragments through adouble stranded piece of DNA, as described in, e.g., U.S. Pat. No.5,635,602; bispecific fusion proteins, e.g., an expression constructcontaining two scFvs with a hydrophilic helical peptide linker betweenthem and a full constant region, as described in, e.g., U.S. Pat. No.5,637,481; multivalent and multispecific binding proteins, e.g., dimerof polypeptides having first domain with binding region of Ig heavychain variable region, and second domain with binding region of Ig lightchain variable region, generally termed diabodies (higher orderstructures are also encompassed creating for bispecific, trispecific, ortetraspecific molecules, as described in, e.g., U.S. Pat. No. 5,837,242;minibody constructs with linked VL and VH chains further connected withpeptide spacers to an antibody hinge region and CH3 region, which can bedimerized to form bispecific/multivalent molecules, as described in,e.g., U.S. Pat. No. 5,837,821; VH and VL domains linked with a shortpeptide linker (e.g., 5 or 10 amino acids) or no linker at all in eitherorientation, which can form dimers to form bispecific diabodies; trimersand tetramers, as described in, e.g., U.S. Pat. No. 5,844,094; String ofVH domains (or VL domains in family members) connected by peptidelinkages with crosslinkable groups at the C-terminus further associatedwith VL domains to form a series of FVs (or scFvs), as described in,e.g., U.S. Pat. No. 5,864,019; and single chain binding polypeptideswith both a VH and a VL domain linked through a peptide linker arecombined into multivalent structures through non-covalent or chemicalcrosslinking to form, e.g., homobivalent, heterobivalent, trivalent, andtetravalent structures using both scFV or diabody type format, asdescribed in, e.g., U.S. Pat. No. 5,869,620. Additional exemplarymultispecific and bispecific molecules and methods of making the sameare found, for example, in U.S. Pat. Nos. 5,910,573, 5,932,448,5,959,083, 5,989,830, 6,005,079, 6,239,259, 6,294,353, U.S. Pat. Nos.6,333,396, 6,476,198, 6,511,663, 6,670,453, 6,743,896, 6,809,185,6,833,441, 7,129,330, 7,183,076, 7,521,056, 7,527,787, 7,534,866,7,612,181, US2002004587A1, US2002076406A1, US2002103345A1,US2003207346A1, US2003211078A1, US2004219643A1, US2004220388A1,US2004242847A1, US2005003403A1, US2005004352A1, US2005069552A1,US2005079170A1, US2005100543A1, US2005136049A1, US2005136051A1,US2005163782A1, US2005266425A1, US2006083747A1, US2006120960A1,US2006204493A1, US2006263367A1, US2007004909A1, US2007087381A1,US2007128150A1, US2007141049A1, US2007154901A1, US2007274985A1,US2008050370A1, US2008069820A1, US2008152645A1, US2008171855A1,US2008241884A1, US2008254512A1, US2008260738A1, US2009130106A1,US2009148905A1, US2009155275A1, US2009162359A1, US2009162360A1,US2009175851A1, US2009175867A1, US2009232811A1, US2009234105A1,US2009263392A1, US2009274649A1, EP346087A2, WO0006605A2, WO02072635A2,WO04081051A1, WO06020258A2, WO2007044887A2, WO2007095338A2,WO2007137760A2, WO2008119353A1, WO2009021754A2, WO2009068630A1,WO9103493A1, WO9323537A1, WO9409131A1, WO9412625A2, WO9509917A1,WO9637621A2, WO9964460A1. The contents of the above-referencedapplications are incorporated herein by reference in their entireties.

Within each antibody or antibody fragment (e.g., scFv) of a bispecificantibody molecule, the VH can be upstream or downstream of the VL. Insome embodiments, the upstream antibody or antibody fragment (e.g.,scFv) is arranged with its VH (VH₁) upstream of its VL (VL₁) and thedownstream antibody or antibody fragment (e.g., scFv) is arranged withits VL (VL₂) upstream of its VH (VH₂), such that the overall bispecificantibody molecule has the arrangement VH₁-VL₁-VL₂-VH₂. In otherembodiments, the upstream antibody or antibody fragment (e.g., scFv) isarranged with its VL (VL₁) upstream of its VH (VH₁) and the downstreamantibody or antibody fragment (e.g., scFv) is arranged with its VH (VH₂)upstream of its VL (VL₂), such that the overall bispecific antibodymolecule has the arrangement VL₁-VH₁-VH₂-VL₂. Optionally, a linker isdisposed between the two antibodies or antibody fragments (e.g., scFvs),e.g., between VL₁ and VL₂ if the construct is arranged asVH₁-VL₁-VL₂-VH₂, or between VH₁ and VH₂ if the construct is arranged asVL₁-VH₁-VH₂-VL₂. The linker may be a linker as described herein, e.g., a(Gly₄-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 4 (SEQID NO: 26). In general, the linker between the two scFvs should be longenough to avoid mispairing between the domains of the two scFvs.Optionally, a linker is disposed between the VL and VH of the firstscFv. Optionally, a linker is disposed between the VL and VH of thesecond scFv. In constructs that have multiple linkers, any two or moreof the linkers can be the same or different. Accordingly, in someembodiments, a bispecific CAR comprises VLs, VHs, and optionally one ormore linkers in an arrangement as described herein.

In one aspect, the bispecific antibody molecule is characterized by afirst immunoglobulin variable domain sequence, e.g., a scFv, which hasbinding specificity for CD33, e.g., comprises a scFv as describedherein, e.g., as described in Table 2 or Table 9, or comprises the lightchain CDRs and/or heavy chain CDRs from a CD33 scFv described herein,and a second immunoglobulin variable domain sequence that has bindingspecificity for a second epitope on a different antigen. In some aspectsthe second immunoglobulin variable domain sequence has bindingspecificity for an antigen expressed on AML cells, e.g., an antigenother than CD33. For example, the second immunoglobulin variable domainsequence has binding specificity for CD123. As another example, thesecond immunoglobulin variable domain sequence has binding specificityfor CLL-1. As another example, the second immunoglobulin variable domainsequence has binding specificity for CD34. As another example, thesecond immunoglobulin variable domain sequence has binding specificityfor FLT3. For example, the second immunoglobulin variable domainsequence has binding specificity for folate receptor beta. In someaspects, the second immunoglobulin variable domain sequence has bindingspecificity for an antigen expressed on B-cells, for example, CD19,CD20, CD22 or ROR1.

Chimeric TCR

In one aspect, the CD33 antibodies and antibody fragments of the presentinvention (for example, those disclosed in Tables 2 and 9) can begrafted to one or more constant domain of a T cell receptor (“TCR”)chain, for example, a TCR alpha or TCR beta chain, to create an chimericTCR that binds specificity to CD33. Without being bound by theory, it isbelieved that chimeric TCRs will signal through the TCR complex uponantigen binding. For example, a CD33 scFv as disclosed herein, can begrafted to the constant domain, e.g., at least a portion of theextracellular constant domain, the transmembrane domain and thecytoplasmic domain, of a TCR chain, for example, the TCR alpha chainand/or the TCR beta chain. As another example, a CD33 antibody fragment,for example a VL domain as described herein, can be grafted to theconstant domain of a TCR alpha chain, and a CD33 antibody fragment, forexample a VH domain as described herein, can be grafted to the constantdomain of a TCR beta chain (or alternatively, a VL domain may be graftedto the constant domain of the TCR beta chain and a VH domain may begrafted to a TCR alpha chain). As another example, the CDRs of a CD33antibody or antibody fragment, e.g., the CDRs of a CD33 antibody orantibody fragment as described in Tables 3, 4, 10, 11, 12 or 13 may begrafted into a TCR alpha and/or beta chain to create a chimeric TCR thatbinds specifically to CD33. For example, the LCDRs disclosed herein maybe grafted into the variable domain of a TCR alpha chain and the HCDRsdisclosed herein may be grafted to the variable domain of a TCR betachain, or vice versa. Such chimeric TCRs may be produced by methodsknown in the art (For example, Willemsen R A et al, Gene Therapy 2000;7: 1369-1377; Zhang T et al, Cancer Gene Ther 2004; 11: 487-496; Aggenet al, Gene Ther. 2012 April; 19(4):365-74).

Transmembrane Domain

With respect to the transmembrane domain, in various embodiments, a CARcan be designed to comprise a transmembrane domain that is attached tothe extracellular domain of the CAR. A transmembrane domain can includeone or more additional amino acids adjacent to the transmembrane region,e.g., one or more amino acid associated with the extracellular region ofthe protein from which the transmembrane was derived (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region)and/or one or more additional amino acids associated with theintracellular region of the protein from which the transmembrane proteinis derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids ofthe intracellular region). In one aspect, the transmembrane domain isone that is associated with one of the other domains of the CAR is used.In some instances, the transmembrane domain can be selected or modifiedby amino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membraneproteins, e.g., to minimize interactions with other members of thereceptor complex. In one aspect, the transmembrane domain is capable ofhomodimerization with another CAR on the CAR-expressing cell, e.g., CARTcell, surface. In a different aspect the amino acid sequence of thetransmembrane domain may be modified or substituted so as to minimizeinteractions with the binding domains of the native binding partnerpresent in the same CAR-expressing cell, e.g., CART.

The transmembrane domain may be derived either from a natural or from arecombinant source. Where the source is natural, the domain may bederived from any membrane-bound or transmembrane protein. In one aspectthe transmembrane domain is capable of signaling to the intracellulardomain(s) whenever the CAR has bound to a target. A transmembrane domainof particular use in this invention may include at least thetransmembrane region(s) of e.g., the alpha, beta or zeta chain of theT-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134,CD137, CD154. In some embodiments, a transmembrane domain may include atleast the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27,LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR,HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19,IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D,ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1,ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7,TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG(CD162), LTBR, PAG/Cbp, NKG2D, NKG2C.

In some instances, the transmembrane domain can be attached to theextracellular region of the CAR, e.g., the antigen binding domain of theCAR, via a hinge, e.g., a hinge from a human protein. For example, inone embodiment, the hinge can be a human Ig (immunoglobulin) hinge,e.g., an IgG4 hinge, or a CD8a hinge. In one embodiment, the hinge orspacer comprises (e.g., consists of) the amino acid sequence of SEQ IDNO:2. In one aspect, the transmembrane domain comprises (e.g., consistsof) a transmembrane domain of SEQ ID NO: 6.

In one aspect, the hinge or spacer comprises an IgG4 hinge. For example,in one embodiment, the hinge or spacer comprises a hinge of the aminoacid sequence

(SEQ ID NO: 3) ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM.In some embodiments, the hinge or spacer comprises a hinge encoded by anucleotide sequence of

(SEQ ID NO: 14) GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG.

In one aspect, the hinge or spacer comprises an IgD hinge. For example,in one embodiment, the hinge or spacer comprises a hinge of the aminoacid sequence

(SEQ ID NO: 4) RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH.In some embodiments, the hinge or spacer comprises a hinge encoded by anucleotide sequence of

(SEQ ID NO: 15) AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT.

In one aspect, the transmembrane domain may be recombinant, in whichcase it will comprise predominantly hydrophobic residues such as leucineand valine. In one aspect a triplet of phenylalanine, tryptophan andvaline can be found at each end of a recombinant transmembrane domain.

Optionally, a short oligo- or polypeptide linker, between 2 and 10 aminoacids in length may form the linkage between the transmembrane domainand the cytoplasmic region of the CAR. A glycine-serine doublet providesa particularly suitable linker. For example, in one aspect, the linkercomprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:5). In someembodiments, the linker is encoded by a nucleotide sequence of

(SEQ ID NO : 16) GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC.

In one aspect, the hinge or spacer comprises a KIR2DS2 hinge.

Cytoplasmic Domain

The cytoplasmic domain or region of the present CAR includes anintracellular signaling domain. An intracellular signaling domain iscapable of activation of at least one of the normal effector functionsof the immune cell in which the CAR has been introduced.

Examples of intracellular signaling domains for use in the CAR of theinvention include the cytoplasmic sequences of the T cell receptor (TCR)and co-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any recombinant sequence that has thesame functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondaryand/or costimulatory signal is also required. Thus, T cell activationcan be said to be mediated by two distinct classes of cytoplasmicsignaling sequences: those that initiate antigen-dependent primaryactivation through the TCR (primary intracellular signaling domains) andthose that act in an antigen-independent manner to provide a secondaryor costimulatory signal (secondary cytoplasmic domain, e.g., acostimulatory domain).

A primary signaling domain regulates primary activation of the TCRcomplex either in a stimulatory way, or in an inhibitory way. Primaryintracellular signaling domains that act in a stimulatory manner maycontain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary intracellular signaling domains thatare of particular use in the invention include those of TCR zeta, FcRgamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a,CD79b, CD278 (also known as “ICOS”), FcεRI, DAP10, DAP12, and CD66d. Inone embodiment, a CAR of the invention comprises an intracellularsignaling domain, e.g., a primary signaling domain of CD3-zeta.

In one embodiment, a primary signaling domain comprises a modified ITAMdomain, e.g., a mutated ITAM domain which has altered (e.g., increasedor decreased) activity as compared to the native ITAM domain. In oneembodiment, a primary signaling domain comprises a modifiedITAM-containing primary intracellular signaling domain, e.g., anoptimized and/or truncated ITAM-containing primary intracellularsignaling domain. In an embodiment, a primary signaling domain comprisesone, two, three, four or more ITAM motifs.

Further examples of molecules containing a primary intracellularsignaling domain that are of particular use in the invention includethose of DAP10, DAP12, and CD32.

The intracellular signalling domain of the CAR can comprise the primarysignalling domain, e.g., CD3-zeta signaling domain, by itself or it canbe combined with any other desired intracellular signaling domain(s)useful in the context of a CAR of the invention. For example, theintracellular signaling domain of the CAR can comprise a primarysignalling domain, e.g., CD3 zeta chain portion, and a costimulatorysignaling domain. The costimulatory signaling domain refers to a portionof the CAR comprising the intracellular domain of a costimulatorymolecule. A costimulatory molecule is a cell surface molecule other thanan antigen receptor or its ligands that is required for an efficientresponse of lymphocytes to an antigen. Examples of such moleculesinclude MHC class I molecule, TNF receptor proteins, Immunoglobulin-likeproteins, cytokine receptors, integrins, signaling lymphocyticactivation molecules (SLAM proteins), activating NK cell receptors,BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40,CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CD5, ICAM-1, ICOS(CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta,IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6,VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM,CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D,NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84,CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3),BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a,and a ligand that specifically binds with CD83, and the like. Forexample, CD27 costimulation has been demonstrated to enhance expansion,effector function, and survival of human CART cells in vitro andaugments human T cell persistence and antitumor activity in vivo (Songet al. Blood. 2012; 119(3):696-706).

The intracellular signaling sequences within the cytoplasmic portion ofthe CAR of the invention may be linked to each other in a random orspecified order. Optionally, a short oligo- or polypeptide linker, forexample, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acids) in length may form the linkage between intracellularsignaling sequence. In one embodiment, a glycine-serine doublet can beused as a suitable linker. In one embodiment, a single amino acid, e.g.,an alanine, a glycine, can be used as a suitable linker.

In one aspect, the intracellular signaling domain is designed tocomprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signalingdomains. In an embodiment, the two or more, e.g., 2, 3, 4, 5, or more,costimulatory signaling domains, are separated by a linker molecule,e.g., a linker molecule described herein. In one embodiment, theintracellular signaling domain comprises two costimulatory signalingdomains. In some embodiments, the linker molecule is a glycine residue.In some embodiments, the linker is an alanine residue.

In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD28. In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain of4-1BB. In one aspect, the signaling domain of 4-1BB is a signalingdomain of SEQ ID NO: 7. In one aspect, the signaling domain of CD3-zetais a signaling domain of SEQ ID NO: 9 (mutant CD3-zeta) or SEQ ID NO: 10(wild type human CD3-zeta).

In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD27. In one aspect, the signaling domain of CD27 comprises an aminoacid sequence of

(SEQ ID NO: 8) QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP.

In one aspect, the signalling domain of CD27 is encoded by a nucleicacid sequence of

(SEQ ID NO: 19) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC.

In one aspect, the intracellular is designed to comprise the signalingdomain of CD3-zeta and the signaling domain of CD28. In one aspect, thesignaling domain of CD28 comprises an amino acid sequence of SEQ ID NO:379. In one aspect, the signaling domain of CD28 is encoded by a nucleicacid sequence of SEQ ID NO: 380.

In one aspect, the intracellular is designed to comprise the signalingdomain of CD3-zeta and the signaling domain of ICOS. In one aspect, thesignaling domain of CD28 comprises an amino acid sequence of SEQ ID NO:381. In one aspect, the signaling domain of ICOS is encoded by a nucleicacid sequence of SEQ ID NO: 382.

In one aspect, the CAR-expressing cell described herein can furthercomprise a second CAR, e.g., a second CAR that includes a differentantigen binding domain, e.g., to the same target (CD33) or a differenttarget (e.g., CD123, CLL-1, CD34, FLT3, or folate receptor beta). In oneembodiment, the second CAR includes an antigen binding domain to atarget expressed on acute myeloid leukemia cells, such as, e.g., CD123,CLL-1, CD34, FLT3, or folate receptor beta. In one embodiment, theCAR-expressing cell comprises a first CAR that targets a first antigenand includes an intracellular signaling domain having a costimulatorysignaling domain but not a primary signaling domain, and a second CARthat targets a second, different, antigen and includes an intracellularsignaling domain having a primary signaling domain but not acostimulatory signaling domain. While not wishing to be bound by theory,placement of a costimulatory signaling domain, e.g., 4-1BB, CD28, CD27,ICOS, or OX-40, onto the first CAR, and the primary signaling domain,e.g., CD3 zeta, on the second CAR can limit the CAR activity to cellswhere both targets are expressed. In one embodiment, the CAR expressingcell comprises a first CD33 CAR that includes a CD33 binding domain, atransmembrane domain and a costimulatory domain and a second CAR thattargets an antigen other than CD33 (e.g., an antigen expressed on AMLcells, e.g., CD123, CLL-1, CD34, FLT3, or folate receptor beta) andincludes an antigen binding domain, a transmembrane domain and a primarysignaling domain. In another embodiment, the CAR expressing cellcomprises a first CD33 CAR that includes a CD33 binding domain, atransmembrane domain and a primary signaling domain and a second CARthat targets an antigen other than CD33 (e.g., an antigen expressed onAML cells, e.g., CD123, CLL-1, CD34, FLT3, or folate receptor beta) andincludes an antigen binding domain to the antigen, a transmembranedomain and a costimulatory signaling domain.

In one embodiment, the CAR-expressing cell comprises a CD33 CARdescribed herein and an inhibitory CAR. In one embodiment, theinhibitory CAR comprises an antigen binding domain that binds an antigenfound on normal cells but not cancer cells, e.g., normal cells that alsoexpress CD33. In one embodiment, the inhibitory CAR comprises theantigen binding domain, a transmembrane domain and an intracellulardomain of an inhibitory molecule. For example, the intracellular domainof the inhibitory CAR can be an intracellular domain of PD1, PD-L1,PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5),LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276),B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHCclass II, GAL9, adenosine, and TGFR beta.

In one embodiment, when the CAR-expressing cell comprises two or moredifferent CARs, the antigen binding domains of the different CARs can besuch that the antigen binding domains do not interact with one another.For example, a cell expressing a first and second CAR can have anantigen binding domain of the first CAR, e.g., as a fragment, e.g., anscFv, that does not form an association with the antigen binding domainof the second CAR, e.g., the antigen binding domain of the second CAR isa VHH.

In some embodiments, the antigen binding domain comprises a singledomain antigen binding (SDAB) molecules include molecules whosecomplementary determining regions are part of a single domainpolypeptide. Examples include, but are not limited to, heavy chainvariable domains, binding molecules naturally devoid of light chains,single domains derived from conventional 4-chain antibodies, engineereddomains and single domain scaffolds other than those derived fromantibodies. SDAB molecules may be any of the art, or any future singledomain molecules. SDAB molecules may be derived from any speciesincluding, but not limited to mouse, human, camel, llama, lamprey, fish,shark, goat, rabbit, and bovine. This term also includes naturallyoccurring single domain antibody molecules from species other thanCamelidae and sharks.

In one aspect, an SDAB molecule can be derived from a variable region ofthe immunoglobulin found in fish, such as, for example, that which isderived from the immunoglobulin isotype known as Novel Antigen Receptor(NAR) found in the serum of shark. Methods of producing single domainmolecules derived from a variable region of NAR (“IgNARs”) are describedin WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

According to another aspect, an SDAB molecule is a naturally occurringsingle domain antigen binding molecule known as heavy chain devoid oflight chains. Such single domain molecules are disclosed in WO 9404678and Hamers-Casterman, C. et al. (1993) Nature 363:446-448, for example.For clarity reasons, this variable domain derived from a heavy chainmolecule naturally devoid of light chain is known herein as a VHH ornanobody to distinguish it from the conventional VH of four chainimmunoglobulins. Such a VHH molecule can be derived from Camelidaespecies, for example in camel, llama, dromedary, alpaca and guanaco.Other species besides Camelidae may produce heavy chain moleculesnaturally devoid of light chain; such VHHs are within the scope of theinvention.

The SDAB molecules can be recombinant, CDR-grafted, humanized,camelized, de-immunized and/or in vitro generated (e.g., selected byphage display).

It has also been discovered, that cells having a plurality of chimericmembrane embedded receptors comprising an antigen binding domain thatinteractions between the antigen binding domain of the receptors can beundesirable, e.g., because it inhibits the ability of one or more of theantigen binding domains to bind its cognate antigen. Accordingly,disclosed herein are cells having a first and a second non-naturallyoccurring chimeric membrane embedded receptor comprising antigen bindingdomains that minimize such interactions. Also disclosed herein arenucleic acids encoding a first and a second non-naturally occurringchimeric membrane embedded receptor comprising a antigen binding domainsthat minimize such interactions, as well as methods of making and usingsuch cells and nucleic acids. In an embodiment the antigen bindingdomain of one of said first said second non-naturally occurring chimericmembrane embedded receptor, comprises an scFv, and the other comprises asingle VH domain, e.g., a camelid, shark, or lamprey single VH domain,or a single VH domain derived from a human or mouse sequence.

In some embodiments, the claimed invention comprises a first and secondCAR, wherein the antigen binding domain of one of said first CAR saidsecond CAR does not comprise a variable light domain and a variableheavy domain. In some embodiments, the antigen binding domain of one ofsaid first CAR said second CAR is an scFv, and the other is not an scFv.In some embodiments, the antigen binding domain of one of said first CARsaid second CAR comprises a single VH domain, e.g., a camelid, shark, orlamprey single VH domain, or a single VH domain derived from a human ormouse sequence. In some embodiments, the antigen binding domain of oneof said first CAR said second CAR comprises a nanobody. In someembodiments, the antigen binding domain of one of said first CAR saidsecond CAR comprises a camelid VHH domain.

In some embodiments, the antigen binding domain of one of said first CARsaid second CAR comprises an scFv, and the other comprises a single VHdomain, e.g., a camelid, shark, or lamprey single VH domain, or a singleVH domain derived from a human or mouse sequence. In some embodiments,the antigen binding domain of one of said first CAR said second CARcomprises an scFv, and the other comprises a nanobody. In someembodiments, the antigen binding domain of one of said first CAR saidsecond CAR comprises comprises an scFv, and the other comprises acamelid VHH domain.

In some embodiments, when present on the surface of a cell, binding ofthe antigen binding domain of said first CAR to its cognate antigen isnot substantially reduced by the presence of said second CAR. In someembodiments, binding of the antigen binding domain of said first CAR toits cognate antigen in the presence of said second CAR is 85%, 90%, 95%,96%, 97%, 98% or 99% of binding of the antigen binding domain of saidfirst CAR to its cognate antigen in the absence of said second CAR.

In some embodiments, when present on the surface of a cell, the antigenbinding domains of said first CAR said second CAR, associate with oneanother less than if both were scFv antigen binding domains. In someembodiments, the antigen binding domains of said first CAR said secondCAR, associate with one another 85%, 90%, 95%, 96%, 97%, 98% or 99% lessthan if both were scFv antigen binding domains.

In another aspect, the CAR-expressing cell described herein can furtherexpress another agent, e.g., an agent which enhances the activity of aCAR-expressing cell. For example, in one embodiment, the agent can be anagent which inhibits an inhibitory molecule. Inhibitory molecules, e.g.,PD1, can, in some embodiments, decrease the ability of a CAR-expressingcell to mount an immune effector response. Examples of inhibitorymolecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g.,CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 orCD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFRbeta. In one embodiment, the agent which inhibits an inhibitorymolecule, e.g., is a molecule described herein, e.g., an agent thatcomprises a first polypeptide, e.g., an inhibitory molecule, associatedwith a second polypeptide that provides a positive signal to the cell,e.g., an intracellular signaling domain described herein. In oneembodiment, the agent comprises a first polypeptide, e.g., of aninhibitory molecule such as PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM(e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT,LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM(TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9,adenosine, and TGFR beta, or a fragment of any of these (e.g., at leasta portion of an extracellular domain of any of these), and a secondpolypeptide which is an intracellular signaling domain described herein(e.g., comprising a costimulatory domain (e.g., 41BB, CD27, ICOS, orCD28, e.g., as described herein) and/or a primary signaling domain(e.g., a CD3 zeta signaling domain described herein). In one embodiment,the agent comprises a first polypeptide of PD1 or a fragment thereof(e.g., at least a portion of an extracellular domain of PD1), and asecond polypeptide of an intracellular signaling domain described herein(e.g., a CD28 signaling domain described herein and/or a CD3 zetasignaling domain described herein). In embodiments, the CAR-expressingcell described herein comprises a switch costimulatory receptor, e.g.,as described in WO 2013/019615, which is incorporated herein byreference in its entirety. PD1 is an inhibitory member of the CD28family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA.PD-1 is expressed on activated B cells, T cells and myeloid cells (Agataet al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2have been shown to downregulate T cell activation upon binding to PD1(Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 NatImmunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 isabundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank etal. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 ClinCancer Res 10:5094). Immune suppression can be reversed by inhibitingthe local interaction of PD1 with PD-L1.

In one embodiment, the agent comprises the extracellular domain (ECD) ofan inhibitory molecule, e.g., Programmed Death 1 (PD1), can be fused toa transmembrane domain and intracellular signaling domains such as 41BBand CD3 zeta (also referred to herein as a PD1 CAR). In one embodiment,the PD1 CAR, when used in combinations with a CD33 CAR described herein,improves the persistence of the CAR-expressing cell, e.g., T or NK cell.In one embodiment, the CAR is a PD1 CAR comprising the extracellulardomain of PD1 indicated as underlined in SEQ ID NO: 24. In oneembodiment, the PD1 CAR comprises the amino acid sequence of SEQ IDNO:24.

(SEQ ID NO: 24) Malpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr.

In one embodiment, the PD1 CAR comprises the amino acid sequenceprovided below (SEQ ID NO:22).

(SEQ ID NO: 22) pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglsta tkdtydalhmqalppr.

In one embodiment, the agent comprises a nucleic acid sequence encodingthe PD1 CAR, e.g., the PD1 CAR described herein. In one embodiment, thenucleic acid sequence for the PD1 CAR is shown below, with the PD1 ECDunderlined below in SEQ ID NO: 23

(SEQ ID NO: 23) atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagaccacccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtcacgaccactccggcgccgcgcccaccgactccggccccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccggaggtgctgtgcatacccggggattggacttcgcatgcgacatctacatttgggctcctctcgccggaacttgtggcgtgctccttctgtccctggtcatcaccctgtactgcaagcggggtcggaaaaagcttctgtacattttcaagcagcccttcatgaggcccgtgcaaaccacccaggaggaggacggttgctcctgccggttccccgaagaggaagaaggaggttgcgagctgcgcgtgaagttctcccggagcgccgacgcccccgcctataagcagggccagaaccagctgtacaacgaactgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctgcagaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatacgatgccctgcacatgcaggccctt ccccctcgc.

In another aspect, the present invention provides a population ofCAR-expressing cells, e.g., CART cells or CAR-expressing NK cells. Insome embodiments, the population of CAR-expressing cells comprises amixture of cells expressing different CARs. For example, in oneembodiment, the population of CAR-expressing cells (e.g., CART cells orCAR-expressing NK cells) can include a first cell expressing a CARhaving a CD33 binding domain described herein, and a second cellexpressing a CAR having a different CD33 binding domain, e.g., a CD33binding domain described herein that differs from the CD33 bindingdomain in the CAR expressed by the first cell. As another example, thepopulation of CAR-expressing cells can include a first cell expressing aCAR that includes a CD33 binding domain, e.g., as described herein, anda second cell expressing a CAR that includes an antigen binding domainto a target other than CD33 (e.g., CD123, CD34, CLL-1, FLT3, or folatereceptor beta). In one embodiment, the population of CAR-expressingcells includes, e.g., a first cell expressing a CAR that includes aprimary intracellular signaling domain, and a second cell expressing aCAR that includes a secondary signaling domain, e.g., a costimulatorysignaling domain.

In another aspect, the present invention provides a population of cellswherein at least one cell in the population expresses a CAR having aCD33 domain described herein, and a second cell expressing anotheragent, e.g., an agent which enhances the activity of a CAR-expressingcell. For example, in one embodiment, the agent can be an agent whichinhibits an inhibitory molecule. Inhibitory molecules, e.g., can, insome embodiments, decrease the ability of a CAR-expressing cell to mountan immune effector response. Examples of inhibitory molecules includePD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, GAL9, adenosine, and TGFR beta. In oneembodiment, the agent which inhibits an inhibitory molecule, e.g., isdescribed herein, e.g., the agent comprises a first polypeptide, e.g.,an inhibitory molecule, associated with a second polypeptide thatprovides a positive signal to the cell, e.g., an intracellular signalingdomain described herein. In one embodiment, the agent comprises a firstpolypeptide, e.g., of an inhibitory molecule such as PD1, PD-L1, PD-L2,CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3,VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II,GAL9, adenosine, and TGFR beta, or a fragment of any of these (e.g., atleast a portion of an extracellular domain of any of these), and asecond polypeptide which is an intracellular signaling domain describedherein (e.g., comprising a costimulatory domain (e.g., 41BB, CD27, ICOS,or CD28, e.g., as described herein) and/or a primary signaling domain(e.g., a CD3 zeta signaling domain described herein). In one embodiment,the agent comprises a first polypeptide of PD1 or a fragment thereof(e.g., at least a portion of the extracellular domain of PD1), and asecond polypeptide of an intracellular signaling domain described herein(e.g., a CD28 signaling domain described herein and/or a CD3 zetasignaling domain described herein).

In one aspect, the present invention provides methods comprisingadministering a population of CAR-expressing cells (e.g., CART cells orCAR-expressing NK cells), e.g., a mixture of cells expressing differentCARs, in combination with another agent, e.g., a kinase inhibitor, suchas a kinase inhibitor described herein. In another aspect, the presentinvention provides methods comprising administering a population ofcells wherein at least one cell in the population expresses a CAR havingan anti-cancer associated antigen binding domain as described herein,and a second cell expressing another agent, e.g., an agent whichenhances the activity of a CAR-expressing cell, in combination withanother agent, e.g., a kinase inhibitor, such as a kinase inhibitordescribed herein.

Natural Killer Cell Receptor (NKR) CARs

In an embodiment, the CAR molecule described herein comprises one ormore components of a natural killer cell receptor (NKR), thereby formingan NKR-CAR. The NKR component can be a transmembrane domain, a hingedomain, or a cytoplasmic domain from any of the following natural killercell receptors: killer cell immunoglobulin-like receptor (KIR), e.g.,KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2,KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, andKIR3DP1; natural cyotoxicity receptor (NCR), e.g., NKp30, NKp44, NKp46;signaling lymphocyte activation molecule (SLAM) family of immune cellreceptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, andCD2F-10; Fc receptor (FcR), e.g., CD16, and CD64; and Ly49 receptors,e.g., LY49A, LY49C. The NKR-CAR molecules described herein may interactwith an adaptor molecule or intracellular signaling domain, e.g., DAP12.Exemplary configurations and sequences of CAR molecules comprising NKRcomponents are described in International Publication No. WO2014/145252,the contents of which are hereby incorporated by reference.

Strategies for Regulating Chimeric Antigen Receptors

There are many ways CAR activities can be regulated. In someembodiments, a regulatable CAR (RCAR) where the CAR activity can becontrolled is desirable to optimize the safety and efficacy of a CARtherapy. For example, inducing apoptosis using, e.g., a caspase fused toa dimerization domain (see, e.g., Di et al., N Engl. J. Med. 2011 Nov.3; 365(18):1673-1683), can be used as a safety switch in the CAR therapyof the instant invention. In another example, CAR-expressing cells canalso express an inducible Caspase-9 (iCaspase-9) molecule that, uponadministration of a dimerizer drug (e.g., rimiducid (also called AP1903(Bellicum Pharmaceuticals) or AP20187 (Ariad)) leads to activation ofthe Caspase-9 and apoptosis of the cells. The iCaspase-9 moleculecontains a chemical inducer of dimerization (CID) binding domain thatmediates dimerization in the presence of a CID. This results ininducible and selective depletion of CAR-expressing cells. In somecases, the iCaspase-9 molecule is encoded by a nucleic acid moleculeseparate from the CAR-encoding vector(s). In some cases, the iCaspase-9molecule is encoded by the same nucleic acid molecule as theCAR-encoding vector. The iCaspase-9 can provide a safety switch to avoidany toxicity of CAR-expressing cells. See, e.g., Song et al. Cancer GeneTher. 2008; 15(10):667-75; Clinical Trial Id. No. NCT02107963; and DiStasi et al. N. Engl. J. Med. 2011; 365:1673-83.

Alternative strategies for regulating the CAR therapy of the instantinvention include utilizing small molecules or antibodies thatdeactivate or turn off CAR activity, e.g., by deleting CAR-expressingcells, e.g., by inducing antibody dependent cell-mediated cytotoxicity(ADCC). For example, CAR-expressing cells described herein may alsoexpress an antigen that is recognized by molecules capable of inducingcell death, e.g., ADCC or compliment-induced cell death. For example,CAR expressing cells described herein may also express a receptorcapable of being targeted by an antibody or antibody fragment. Examplesof such receptors include EpCAM, VEGFR, integrins (e.g., integrins ανβ3,α4, αI3/4β3, α4β7, α5β1, ανβ3, αν), members of the TNF receptorsuperfamily (e.g., TRAIL-R1, TRAIL-R2), PDGF Receptor, interferonreceptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1,TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11,CD11a/LFA-1, CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/lgE Receptor,CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74,CD80, CD125, CD147/basigin, CD152/CTLA-4, CD154/CD40L, CD195/CCR5,CD319/SLAMF7, and EGFR, and truncated versions thereof (e.g., versionspreserving one or more extracellular epitopes but lacking one or moreregions within the cytoplasmic domain). For example, CAR-expressingcells described herein may also express truncated epidermal growthfactor receptor (EGFR) which lacks signaling capacity but retains theepitope that is recognized by molecules capable of inducing ADCC, e.g.,cetuximab (ERBITUX®), such that administration of cetuximab induces ADCCand subsequent depletion of the CAR-expressing cells (see, e.g.,WO2011/056894, and Jonnalagadda et al., Gene Ther. 2013; 20(8)853-860).Another strategy includes expressing a highly compact marker/suicidegene that combines target epitopes from both CD32 and CD20 antigens inthe CAR-expressing cells described herein, which binds rituximab,resulting in selective depletion of the CAR-expressing cells, e.g., byADCC (see, e.g., Philip et al., Blood. 2014; 124(8)1277-1287). Othermethods for depleting CAR-expressing cells described herein includeadministration of CAMPATH®, a monoclonal anti-CD52 antibody thatselectively binds and targets mature lymphocytes, e.g., CAR-expressingcells, for destruction, e.g., by inducing ADCC. In other embodiments,CAR-expressing cells can be selectively targeted using a CAR ligand,e.g., an anti-idiotypic antibody. In some embodiments, theanti-idiotypic antibody can cause effector cell activity, e.g., ADCC orADC activities, thereby reducing the number of CAR-expressing cells. Inother embodiments, the CAR ligand, e.g., the anti-idiotypic antibody,can be coupled to an agent that induces cell killing, e.g., a toxin,thereby reducing the number of CAR-expressing cells. Alternatively, theCAR molecules themselves can be configured such that the activity can beregulated, e.g., turned on and off, as described below.

In some embodiments, a RCAR comprises a set of polypeptides, typicallytwo in the simplest embodiments, in which the components of a standardCAR described herein, e.g., an antigen binding domain and anintracellular signaling domain, are partitioned on separate polypeptidesor members. In some embodiments, the set of polypeptides include adimerization switch that, upon the presence of a dimerization molecule,can couple the polypeptides to one another, e.g., can couple an antigenbinding domain to an intracellular signaling domain. Additionaldescription and exemplary configurations of such regulatable CARs areprovided herein and in International Publication No. WO 2015/090229,hereby incorporated by reference in its entirety.

In an embodiment, an RCAR comprises two polypeptides or members: 1) anintracellular signaling member comprising an intracellular signalingdomain, e.g., a primary intracellular signaling domain described herein,and a first switch domain; 2) an antigen binding member comprising anantigen binding domain, e.g., that targets a tumor antigen describedherein, as described herein and a second switch domain. Optionally, theRCAR comprises a transmembrane domain described herein. In anembodiment, a transmembrane domain can be disposed on the intracellularsignaling member, on the antigen binding member, or on both. (Unlessotherwise indicated, when members or elements of an RCAR are describedherein, the order can be as provided, but other orders are included aswell. In other words, in an embodiment, the order is as set out in thetext, but in other embodiments, the order can be different. E.g., theorder of elements on one side of a transmembrane region can be differentfrom the example, e.g., the placement of a switch domain relative to aintracellular signaling domain can be different, e.g., reversed).

In an embodiment, the first and second switch domains can form anintracellular or an extracellular dimerization switch. In an embodiment,the dimerization switch can be a homodimerization switch, e.g., wherethe first and second switch domain are the same, or a heterodimerizationswitch, e.g., where the first and second switch domain are differentfrom one another.

In embodiments, an RCAR can comprise a “multi switch.” A multi switchcan comprise heterodimerization switch domains or homodimerizationswitch domains. A multi switch comprises a plurality of, e.g., 2, 3, 4,5, 6, 7, 8, 9, or 10, switch domains, independently, on a first member,e.g., an antigen binding member, and a second member, e.g., anintracellular signaling member. In an embodiment, the first member cancomprise a plurality of first switch domains, e.g., FKBP-based switchdomains, and the second member can comprise a plurality of second switchdomains, e.g., FRB-based switch domains. In an embodiment, the firstmember can comprise a first and a second switch domain, e.g., aFKBP-based switch domain and a FRB-based switch domain, and the secondmember can comprise a first and a second switch domain, e.g., aFKBP-based switch domain and a FRB-based switch domain.

In an embodiment, the intracellular signaling member comprises one ormore intracellular signaling domains, e.g., a primary intracellularsignaling domain and one or more costimulatory signaling domains.

In an embodiment, the antigen binding member may comprise one or moreintracellular signaling domains, e.g., one or more costimulatorysignaling domains. In an embodiment, the antigen binding membercomprises a plurality, e.g., 2 or 3 costimulatory signaling domainsdescribed herein, e.g., selected from 4-1BB, CD28, CD27, ICOS, and OX40,and in embodiments, no primary intracellular signaling domain. In anembodiment, the antigen binding member comprises the followingcostimulatory signaling domains, from the extracellular to intracellulardirection: 4-1BB-CD27; 41BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28-4-1BB;OX40-CD28; CD28-OX40; CD28-4-1BB; or 4-1BB-CD28. In such embodiments,the intracellular binding member comprises a CD3zeta domain. In one suchembodiment the RCAR comprises (1) an antigen binding member comprising,an antigen binding domain, a transmembrane domain, and two costimulatorydomains and a first switch domain; and (2) an intracellular signalingdomain comprising a transmembrane domain or membrane tethering domainand at least one primary intracellular signaling domain, and a secondswitch domain.

An embodiment provides RCARs wherein the antigen binding member is nottethered to the surface of the CAR cell. This allows a cell having anintracellular signaling member to be conveniently paired with one ormore antigen binding domains, without transforming the cell with asequence that encodes the antigen binding member. In such embodiments,the RCAR comprises: 1) an intracellular signaling member comprising: afirst switch domain, a transmembrane domain, an intracellular signalingdomain, e.g., a primary intracellular signaling domain, and a firstswitch domain; and 2) an antigen binding member comprising: an antigenbinding domain, and a second switch domain, wherein the antigen bindingmember does not comprise a transmembrane domain or membrane tetheringdomain, and, optionally, does not comprise an intracellular signalingdomain. In some embodiments, the RCAR may further comprise 3) a secondantigen binding member comprising: a second antigen binding domain,e.g., a second antigen binding domain that binds a different antigenthan is bound by the antigen binding domain; and a second switch domain.

Also provided herein are RCARs wherein the antigen binding membercomprises bispecific activation and targeting capacity. In thisembodiment, the antigen binding member can comprise a plurality, e.g.,2, 3, 4, or 5 antigen binding domains, e.g., scFvs, wherein each antigenbinding domain binds to a target antigen, e.g. different antigens or thesame antigen, e.g., the same or different epitopes on the same antigen.In an embodiment, the plurality of antigen binding domains are intandem, and optionally, a linker or hinge region is disposed betweeneach of the antigen binding domains. Suitable linkers and hinge regionsare described herein.

An embodiment provides RCARs having a configuration that allowsswitching of proliferation. In this embodiment, the RCAR comprises: 1)an intracellular signaling member comprising: optionally, atransmembrane domain or membrane tethering domain; one or moreco-stimulatory signaling domain, e.g., selected from 4-1BB, CD28, CD27,ICOS, and OX40, and a switch domain; and 2) an antigen binding membercomprising: an antigen binding domain, a transmembrane domain, and aprimary intracellular signaling domain, e.g., a CD3zeta domain, whereinthe antigen binding member does not comprise a switch domain, or doesnot comprise a switch domain that dimerizes with a switch domain on theintracellular signaling member. In an embodiment, the antigen bindingmember does not comprise a co-stimulatory signaling domain. In anembodiment, the intracellular signaling member comprises a switch domainfrom a homodimerization switch. In an embodiment, the intracellularsignaling member comprises a first switch domain of a heterodimerizationswitch and the RCAR comprises a second intracellular signaling memberwhich comprises a second switch domain of the heterodimerization switch.In such embodiments, the second intracellular signaling member comprisesthe same intracellular signaling domains as the intracellular signalingmember. In an embodiment, the dimerization switch is intracellular. Inan embodiment, the dimerization switch is extracellular.

In any of the RCAR configurations described here, the first and secondswitch domains comprise a FKBP-FRB based switch as described herein.

Also provided herein are cells comprising an RCAR described herein. Anycell that is engineered to express a RCAR can be used as a RCARX cell.In an embodiment the RCARX cell is a T cell, and is referred to as aRCART cell. In an embodiment the RCARX cell is an NK cell, and isreferred to as a RCARN cell.

Also provided herein are nucleic acids and vectors comprising RCARencoding sequences. Sequence encoding various elements of an RCAR can bedisposed on the same nucleic acid molecule, e.g., the same plasmid orvector, e.g., viral vector, e.g., lentiviral vector. In an embodiment,(i) sequence encoding an antigen binding member and (ii) sequenceencoding an intracellular signaling member, can be present on the samenucleic acid, e.g., vector. Production of the corresponding proteins canbe achieved, e.g., by the use of separate promoters, or by the use of abicistronic transcription product (which can result in the production oftwo proteins by cleavage of a single translation product or by thetranslation of two separate protein products). In an embodiment, asequence encoding a cleavable peptide, e.g., a P2A or F2A sequence, isdisposed between (i) and (ii). In an embodiment, a sequence encoding anIRES, e.g., an EMCV or EV71 IRES, is disposed between (i) and (ii). Inthese embodiments, (i) and (ii) are transcribed as a single RNA. In anembodiment, a first promoter is operably linked to (i) and a secondpromoter is operably linked to (ii), such that (i) and (ii) aretranscribed as separate mRNAs.

Alternatively, the sequence encoding various elements of an RCAR can bedisposed on the different nucleic acid molecules, e.g., differentplasmids or vectors, e.g., viral vector, e.g., lentiviral vector. E.g.,the (i) sequence encoding an antigen binding member can be present on afirst nucleic acid, e.g., a first vector, and the (ii) sequence encodingan intracellular signaling member can be present on the second nucleicacid, e.g., the second vector.

Dimerization Switches

Dimerization switches can be non-covalent or covalent. In a non-covalentdimerization switch, the dimerization molecule promotes a non-covalentinteraction between the switch domains. In a covalent dimerizationswitch, the dimerization molecule promotes a covalent interactionbetween the switch domains.

In an embodiment, the RCAR comprises a FKBP/FRAP, or FKBP/FRB,-baseddimerization switch. FKBP12 (FKBP, or FK506 binding protein) is anabundant cytoplasmic protein that serves as the initial intracellulartarget for the natural product immunosuppressive drug, rapamycin.Rapamycin binds to FKBP and to the large PI3K homolog FRAP (RAFT, mTOR).FRB is a 93 amino acid portion of FRAP, that is sufficient for bindingthe FKBP-rapamycin complex (Chen, J., Zheng, X. F., Brown, E. J. &Schreiber, S. L. (1995) Identification of an 11-kDaFKBP12-rapamycin-binding domain within the 289-kDaFKBP12-rapamycin-associated protein and characterization of a criticalserine residue. Proc Natl Acad Sci USA 92: 4947-51.)

In embodiments, an FKBP/FRAP, e.g., an FKBP/FRB, based switch can use adimerization molecule, e.g., rapamycin or a rapamycin analog.

The amino acid sequence of FKBP is as follows:

(SEQ ID NO: 148) D V P D Y A S L G G P S S P K K K R K V S R G V Q VE T I S P G D G R T F P K R G Q T C V V H Y T G M LE D G K K F D S S R D R N K P F K F M L G K Q E V IR G W E E G V A Q M S V G Q R A K L T I S P D Y A YG A T G H P G I I P P H A T L V F D V E L L K L E T S Y

In embodiments, an FKBP switch domain can comprise a fragment of FKBPhaving the ability to bind with FRB, or a fragment or analog thereof, inthe presence of rapamycin or a rapalog, e.g., the underlined portion ofSEQ ID NO: 148, which is:

(SEQ ID NO: 149) V Q V E T I S P G D G R T F P K R G Q T C V V H Y TG M L E D G K K F D S S R D R N K P F K F M L G K QE V I R G W E E G V A Q M S V G Q R A K L T I S P DY A Y G A T G H P G I I P P H A T L V F D V E L L K L E T S

The amino acid sequence of FRB is as follows:

(SEQ ID NO: 150) ILWHEMWHEG LEEASRLYFG ERNVKGMFEV LEPLHAMMERGPQTLKETSF NQAYGRDLME AQEWCRKYMK SGNVKDLTQA WDLYYHVFRR ISK

“FKBP/FRAP, e.g., an FKBP/FRB, based switch” as that term is usedherein, refers to a dimerization switch comprising: a first switchdomain, which comprises an FKBP fragment or analog thereof having theability to bind with FRB, or a fragment or analog thereof, in thepresence of rapamycin or a rapalog, e.g., RAD001, and has at least 70,75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by nomore than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from,the FKBP sequence of SEQ ID NO: 148 or 149; and a second switch domain,which comprises an FRB fragment or analog thereof having the ability tobind with FRB, or a fragment or analog thereof, in the presence ofrapamycin or a rapalog, and has at least 70, 75, 80, 85, 90, 95, 96, 97,98, or 99% identity with, or differs by no more than 30, 25, 20, 15, 10,5, 4, 3, 2, or 1 amino acid residues from, the FRB sequence of SEQ IDNO: 150. In an embodiment, a RCAR described herein comprises one switchdomain comprises amino acid residues disclosed in SEQ ID NO: 148 (or SEQID NO: 149), and one switch domain comprises amino acid residuesdisclosed in SEQ ID NO: 150.

In embodiments, the FKBP/FRB dimerization switch comprises a modifiedFRB switch domain that exhibits altered, e.g., enhanced, complexformation between an FRB-based switch domain, e.g., the modified FRBswitch domain, a FKBP-based switch domain, and the dimerizationmolecule, e.g., rapamycin or a rapalogue, e.g., RAD001. In anembodiment, the modified FRB switch domain comprises one or moremutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, selected frommutations at amino acid position(s) L2031, E2032, 52035, R2036, F2039,G2040, T2098, W2101, D2102, Y2105, and F2108, where the wild-type aminoacid is mutated to any other naturally-occurring amino acid. In anembodiment, a mutant FRB comprises a mutation at E2032, where E2032 ismutated to phenylalanine (E2032F), methionine (E2032M), arginine(E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E20321), e.g.,SEQ ID NO: 151, or leucine (E2032L), e.g., SEQ ID NO: 152. In anembodiment, a mutant FRB comprises a mutation at T2098, where T2098 ismutated to phenylalanine (T2098F) or leucine (T2098L), e.g., SEQ ID NO:153. In an embodiment, a mutant FRB comprises a mutation at E2032 and atT2098, where E2032 is mutated to any amino acid, and where T2098 ismutated to any amino acid, e.g., SEQ ID NO: 154. In an embodiment, amutant FRB comprises an E20321 and a T2098L mutation, e.g., SEQ ID NO:155. In an embodiment, a mutant FRB comprises an E2032L and a T2098Lmutation, e.g., SEQ ID NO: 156.

TABLE 6Exemplary mutant FRB having increased affinity for a dimerization molecule.SEQ ID FRB mutant Amino Acid Sequence NO: E2032I mutantILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 151DLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS E2032L mutantILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 152DLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS T2098L mutantILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 153DLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS E2032, T2098 ILWHEMWHEGL XEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 154 mutantDLMEAQEWCRKYMKSGNVKDL X QAWDLYYHVFRRISKTS E2032I, T2098LILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 155 mutantDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS E2032L, T2098LILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 156 mutantDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS

Other suitable dimerization switches include a GyrB-GyrB baseddimerization switch, a Gibberellin-based dimerization switch, atag/binder dimerization switch, and a halo-tag/snap-tag dimerizationswitch. Following the guidance provided herein, such switches andrelevant dimerization molecules will be apparent to one of ordinaryskill.

Dimerization Molecule

Association between the switch domains is promoted by the dimerizationmolecule. In the presence of dimerization molecule interaction orassociation between switch domains allows for signal transductionbetween a polypeptide associated with, e.g., fused to, a first switchdomain, and a polypeptide associated with, e.g., fused to, a secondswitch domain. In the presence of non-limiting levels of dimerizationmolecule signal transduction is increased by 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 5, 10, 50, 100 fold, e.g., as measured in asystem described herein.

Rapamycin and rapamycin analogs (sometimes referred to as rapalogues),e.g., RAD001, can be used as dimerization molecules in a FKBP/FRB-baseddimerization switch described herein. In an embodiment the dimerizationmolecule can be selected from rapamycin (sirolimus), RAD001(everolimus), zotarolimus, temsirolimus, AP-23573 (ridaforolimus),biolimus and AP21967. Additional rapamycin analogs suitable for use withFKBP/FRB-based dimerization switches are further described in thesection entitled “Combination Therapies”, or in the subsection entitled“Combination with a Low, Immune Enhancing, Dose of an mTOR inhibitor”.

Split CAR

In some embodiments, the CAR-expressing cell uses a split CAR. The splitCAR approach is described in more detail in publications WO2014/055442and WO2014/055657, incorporated herein by reference. Briefly, a splitCAR system comprises a cell expressing a first CAR having a firstantigen binding domain and a costimulatory domain (e.g., 41BB), and thecell also expresses a second CAR having a second antigen binding domainand an intracellular signaling domain (e.g., CD3 zeta). When the cellencounters the first antigen, the costimulatory domain is activated, andthe cell proliferates. When the cell encounters the second antigen, theintracellular signaling domain is activated and cell-killing activitybegins. Thus, the CAR-expressing cell is only fully activated in thepresence of both antigens. In embodiments the first antigen bindingdomain recognizes CD33, e.g., comprises an antigen binding domaindescribed herein, and the second antigen binding domain recognizes anantigen expressed on acute myeloid leukemia cells, e.g., CD123, CLL-1,CD34, FLT3, or folate receptor beta. In embodiments the first antigenbinding domain recognizes CD33, e.g., comprises an antigen bindingdomain described herein, and the second antigen binding domainrecognizes an antigen expressed on B-cells, e.g., CD19, CD20, CD22 orROR1.

Stability and Mutations

The stability of a CD33 binding domain, e.g., scFv molecules (e.g.,soluble scFv) can be evaluated in reference to the biophysicalproperties (e.g., thermal stability) of a conventional control scFvmolecule or a full length antibody. In one embodiment, the human scFvhas a thermal stability that is greater than about 0.1, about 0.25,about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees,about 13 degrees, about 14 degrees, or about 15 degrees Celsius than acontrol binding molecule (e.g. a conventional scFv molecule) in thedescribed assays.

The improved thermal stability of the CD33 binding domain, e.g., scFv issubsequently conferred to the entire CAR33 construct, leading toimproved therapeutic properties of the CAR33 construct. The thermalstability of the CD33 binding domain, e.g., scFv can be improved by atleast about 2° C. or 3° C. as compared to a conventional antibody. Inone embodiment, the CD33 binding domain, e.g., scFv has a 1° C. improvedthermal stability as compared to a conventional antibody. In anotherembodiment, the CD33 binding domain, e.g., scFv has a 2° C. improvedthermal stability as compared to a conventional antibody. In anotherembodiment, the scFv has a 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15° C.improved thermal stability as compared to a conventional antibody.Comparisons can be made, for example, between the scFv moleculesdisclosed herein and full length antibodies. Thermal stability can bemeasured using methods known in the art. For example, in one embodiment,Tm can be measured. Methods for measuring Tm and other methods ofdetermining protein stability are described in more detail below.

Mutations in scFv alter the stability of the scFv and improve theoverall stability of the scFv and the CAR33 construct. Stability of thehumanized or human scFv is determined using measurements such as Tm,temperature denaturation and temperature aggregation.

The binding capacity of the mutant scFvs can be determined using assaysdescribed in the Examples.

In one embodiment, the CD33 binding domain, e.g., scFv comprises atleast one mutation such that the mutated scFv confers improved stabilityto the CAR33 construct. In another embodiment, the CD33 binding domain,e.g., scFv comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutationsarising from the humanization process such that the mutated scFv confersimproved stability to the CAR33 construct.

Methods of Evaluating Protein Stability

The stability of an antigen binding domain may be assessed using, e.g.,the methods described below. Such methods allow for the determination ofmultiple thermal unfolding transitions where the least stable domaineither unfolds first or limits the overall stability threshold of amultidomain unit that unfolds cooperatively (e.g., a multidomain proteinwhich exhibits a single unfolding transition). The least stable domaincan be identified in a number of additional ways. Mutagenesis can beperformed to probe which domain limits the overall stability.Additionally, protease resistance of a multidomain protein can beperformed under conditions where the least stable domain is known to beintrinsically unfolded via DSC or other spectroscopic methods (Fontana,et al., (1997) Fold. Des., 2: R17-26; Dimasi et al. (2009) J. Mol. Biol.393: 672-692). Once the least stable domain is identified, the sequenceencoding this domain (or a portion thereof) may be employed as a testsequence in the methods.

a) Thermal Stability

The thermal stability of the compositions may be analyzed using a numberof non-limiting biophysical or biochemical techniques known in the art.In certain embodiments, thermal stability is evaluated by analyticalspectroscopy.

An exemplary analytical spectroscopy method is Differential Scanningcalorimetry (DSC). DSC employs a calorimeter which is sensitive to theheat absorbances that accompany the unfolding of most proteins orprotein domains (see, e.g. Sanchez-Ruiz, et al., Biochemistry, 27:1648-52, 1988). To determine the thermal stability of a protein, asample of the protein is inserted into the calorimeter and thetemperature is raised until the Fab or scFv unfolds. The temperature atwhich the protein unfolds is indicative of overall protein stability.

Another exemplary analytical spectroscopy method is Circular Dichroism(CD) spectroscopy. CD spectrometry measures the optical activity of acomposition as a function of increasing temperature. Circular dichroism(CD) spectroscopy measures differences in the absorption of left-handedpolarized light versus right-handed polarized light which arise due tostructural asymmetry. A disordered or unfolded structure results in a CDspectrum very different from that of an ordered or folded structure. TheCD spectrum reflects the sensitivity of the proteins to the denaturingeffects of increasing temperature and is therefore indicative of aproteins thermal stability (see van Mierlo and Steemsma, J. Biotechnol.,79(3):281-98, 2000).

Another exemplary analytical spectroscopy method for measuring thermalstability is Fluorescence Emission Spectroscopy (see van Mierlo andSteemsma, supra). Yet another exemplary analytical spectroscopy methodfor measuring thermal stability is Nuclear Magnetic Resonance (NMR)spectroscopy (see, e.g. van Mierlo and Steemsma, supra).

The thermal stability of a composition can be measured biochemically. Anexemplary biochemical method for assessing thermal stability is athermal challenge assay. In a “thermal challenge assay”, a compositionis subjected to a range of elevated temperatures for a set period oftime. For example, in one embodiment, test scFv molecules or moleculescomprising scFv molecules are subject to a range of increasingtemperatures, e.g., for 1-1.5 hours. The activity of the protein is thenassayed by a relevant biochemical assay. For example, if the protein isa binding protein (e.g. an scFv or scFv-containing polypeptide) thebinding activity of the binding protein may be determined by afunctional or quantitative ELISA.

Such an assay may be done in a high-throughput format and thosedisclosed in the Examples using E. coli and high throughput screening. Alibrary of CD33 binding domains, e.g., scFv variants may be createdusing methods known in the art. CD33 binding domains, e.g., scFvexpression may be induced and the CD33 binding domains, e.g., scFv maybe subjected to thermal challenge. The challenged test samples may beassayed for binding and those CD33 binding domains, e.g., scFvs whichare stable may be scaled up and further characterized.

Thermal stability is evaluated by measuring the melting temperature (Tm)of a composition using any of the above techniques (e.g. analyticalspectroscopy techniques). The melting temperature is the temperature atthe midpoint of a thermal transition curve wherein 50% of molecules of acomposition are in a folded state (See e.g., Dimasi et al. (2009) J. MolBiol. 393: 672-692). In one embodiment, Tm values for a CD33 bindingdomain, e.g., scFv are about 40° C., 41° C., 42° C., 43° C., 44° C., 45°C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54°C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63°C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72°C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81°C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90°C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99°C., 100° C. In one embodiment, Tm values for an IgG is about 40° C., 41°C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50°C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59°C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68°C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77°C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86°C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95°C., 96° C., 97° C., 98° C., 99° C., 100° C. In one embodiment, Tm valuesfor an multivalent antibody is about 40° C., 41° C., 42° C., 43° C., 44°C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53°C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62°C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71°C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80°C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89°C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98°C., 99° C., 100° C.

Thermal stability is also evaluated by measuring the specific heat orheat capacity (Cp) of a composition using an analytical calorimetrictechnique (e.g. DSC). The specific heat of a composition is the energy(e.g. in kcal/mol) is required to rise by 1° C., the temperature of 1mol of water. As large Cp is a hallmark of a denatured or inactiveprotein composition. The change in heat capacity (ΔCp) of a compositionis measured by determining the specific heat of a composition before andafter its thermal transition. Thermal stability may also be evaluated bymeasuring or determining other parameters of thermodynamic stabilityincluding Gibbs free energy of unfolding (ΔG), enthalpy of unfolding(ΔH), or entropy of unfolding (ΔS). One or more of the above biochemicalassays (e.g. a thermal challenge assay) are used to determine thetemperature (i.e. the Tc value) at which 50% of the composition retainsits activity (e.g. binding activity).

In addition, mutations to the CD33 binding domain, e.g., scFv alter thethermal stability of the CD33 binding domain, e.g., scFv compared withthe unmutated CD33 binding domain, e.g., scFv. When the humanized orhuman CD33 binding domain, e.g., scFv is incorporated into a CAR33construct, the CD33 binding domain, e.g., humanized or human scFvconfers thermal stability to the overall CD33 CAR construct. In oneembodiment, the CD33 binding domain, e.g., scFv comprises a singlemutation that confers thermal stability to the CD33 binding domain,e.g., scFv. In another embodiment, the CD33 binding domain, e.g., scFvcomprises multiple mutations that confer thermal stability to the CD33binding domain, e.g., scFv. In one embodiment, the multiple mutations inthe CD33 binding domain, e.g., scFv have an additive effect on thermalstability of the CD33 binding domain, e.g., scFv.

b) % Aggregation

The stability of a composition can be determined by measuring itspropensity to aggregate. Aggregation can be measured by a number ofnon-limiting biochemical or biophysical techniques. For example, theaggregation of a composition may be evaluated using chromatography, e.g.Size-Exclusion Chromatography (SEC). SEC separates molecules on thebasis of size. A column is filled with semi-solid beads of a polymericgel that will admit ions and small molecules into their interior but notlarge ones. When a protein composition is applied to the top of thecolumn, the compact folded proteins (i.e. non-aggregated proteins) aredistributed through a larger volume of solvent than is available to thelarge protein aggregates. Consequently, the large aggregates move morerapidly through the column, and in this way the mixture can be separatedor fractionated into its components. Each fraction can be separatelyquantified (e.g. by light scattering) as it elutes from the gel.Accordingly, the % aggregation of a composition can be determined bycomparing the concentration of a fraction with the total concentrationof protein applied to the gel. Stable compositions elute from the columnas essentially a single fraction and appear as essentially a single peakin the elution profile or chromatogram.

c) Binding Affinity The stability of a composition can be assessed bydetermining its target binding affinity.

A wide variety of methods for determining binding affinity are known inthe art. An exemplary method for determining binding affinity employssurface plasmon resonance. Surface plasmon resonance is an opticalphenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin.51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627; Johnsson, B.,et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al.(1991) Anal. Biochem. 198:268-277.

In one aspect, the antigen binding domain of the CAR comprises an aminoacid sequence that is homologous to an antigen binding domain amino acidsequence described herein, and the antigen binding domain retains thedesired functional properties of the CD33 antibody fragments describedherein. In one specific aspect, the CAR composition of the inventioncomprises an antibody fragment. In a further aspect, that antibodyfragment comprises an scFv.

In various aspects, the antigen binding domain of the CAR is engineeredby modifying one or more amino acids within one or both variable regions(e.g., VH and/or VL), for example within one or more CDR regions and/orwithin one or more framework regions. In one specific aspect, the CARcomposition of the invention comprises an antibody fragment. In afurther aspect, that antibody fragment comprises an scFv.

It will be understood by one of ordinary skill in the art that theantibody or antibody fragment of the invention may further be modifiedsuch that they vary in amino acid sequence (e.g., from wild-type), butnot in desired activity. For example, additional nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues may be made to the protein For example, anonessential amino acid residue in a molecule may be replaced withanother amino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members, e.g., a conservative substitution, in which an aminoacid residue is replaced with an amino acid residue having a similarside chain, may be made.

Families of amino acid residues having similar side chains have beendefined in the art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

Percent identity in the context of two or more nucleic acids orpolypeptide sequences, refers to two or more sequences that are thesame. Two sequences are “substantially identical” if two sequences havea specified percentage of amino acid residues or nucleotides that arethe same (e.g., 60% identity, optionally 70%, 71%. 72%. 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over aspecified region, or, when not specified, over the entire sequence),when compared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Optionally, the identity exists over a region that is atleast about 50 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. Methods of alignment of sequences forcomparison are well known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443, by the search for similarity method of Pearson and Lipman,(1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Brent et al., (2003) Current Protocols inMolecular Biology).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., (1977) Nuc. AcidsRes. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol.215:403-410, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller, (1988)Comput. Appl. Biosci. 4:11-17) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. In addition, the percentidentity between two amino acid sequences can be determined using theNeedleman and Wunsch (1970) J. Mol. Biol. 48:444-453) algorithm whichhas been incorporated into the GAP program in the GCG software package(available at www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

In one aspect, the present invention contemplates modifications of thestarting antibody or fragment (e.g., scFv) amino acid sequence thatgenerate functionally equivalent molecules. For example, the VH or VL ofa CD33 binding domain, e.g., scFv, comprised in the CAR can be modifiedto retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting VH or VLframework region of the CD33 binding domain, e.g., scFv. The presentinvention contemplates modifications of the entire CAR construct, e.g.,modifications in one or more amino acid sequences of the various domainsof the CAR construct in order to generate functionally equivalentmolecules. The CAR construct can be modified to retain at least about70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% identity of the starting CAR construct.

RNA Transfection

Disclosed herein are methods for producing an in vitro transcribed RNACAR. The present invention also includes a CAR encoding RNA constructthat can be directly transfected into a cell. A method for generatingmRNA for use in transfection can involve in vitro transcription (IVT) ofa template with specially designed primers, followed by polyA addition,to produce a construct containing 3′ and 5′ untranslated sequence(“UTR”), a 5′ cap and/or Internal Ribosome Entry Site (IRES), thenucleic acid to be expressed, and a polyA tail, typically 50-2000 basesin length (SEQ ID NO:35). RNA so produced can efficiently transfectdifferent kinds of cells. In one aspect, the template includes sequencesfor the CAR.

In one aspect the CD33 CAR is encoded by a messenger RNA (mRNA). In oneaspect the mRNA encoding the CD33 CAR is introduced into an immuneeffector cell (e.g., T cell or NK cell) for production of aCAR-expressing cell (e.g., CART cell or CAR-expressing NK cell).

In one embodiment, the in vitro transcribed RNA CAR can be introduced toa cell as a form of transient transfection. The RNA is produced by invitro transcription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA can be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA. The desired temple for in vitrotranscription is a CAR of the present invention. For example, thetemplate for the RNA CAR comprises an extracellular region comprising asingle chain variable domain of an anti-tumor antibody; a hinge region,a transmembrane domain (e.g., a transmembrane domain of CD8a); and acytoplasmic region that includes an intracellular signaling domain,e.g., comprising the signaling domain of CD3-zeta and the signalingdomain of 4-1BB.

In one embodiment, the DNA to be used for PCR contains an open readingframe. The DNA can be from a naturally occurring DNA sequence from thegenome of an organism. In one embodiment, the nucleic acid can includesome or all of the 5 and/or 3 untranslated regions (UTRs). The nucleicacid can include exons and introns. In one embodiment, the DNA to beused for PCR is a human nucleic acid sequence. In another embodiment,the DNA to be used for PCR is a human nucleic acid sequence includingthe 5 and 3 UTRs. The DNA can alternatively be an artificial DNAsequence that is not normally expressed in a naturally occurringorganism. An exemplary artificial DNA sequence is one that containsportions of genes that are ligated together to form an open readingframe that encodes a fusion protein. The portions of DNA that areligated together can be from a single organism or from more than oneorganism.

PCR is used to generate a template for in vitro transcription of mRNAwhich is used for transfection. Methods for performing PCR are wellknown in the art. Primers for use in PCR are designed to have regionsthat are substantially complementary to regions of the DNA to be used asa template for the PCR. “Substantially complementary,” as used herein,refers to sequences of nucleotides where a majority or all of the basesin the primer sequence are complementary, or one or more bases arenon-complementary, or mismatched. Substantially complementary sequencesare able to anneal or hybridize with the intended DNA target underannealing conditions used for PCR. The primers can be designed to besubstantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a nucleicacid that is normally transcribed in cells (the open reading frame),including 5 and 3 UTRs. The primers can also be designed to amplify aportion of a nucleic acid that encodes a particular domain of interest.In one embodiment, the primers are designed to amplify the coding regionof a human cDNA, including all or portions of the 5 and 3 UTRs. Primersuseful for PCR can be generated by synthetic methods that are well knownin the art. “Forward primers” are primers that contain a region ofnucleotides that are substantially complementary to nucleotides on theDNA template that are upstream of the DNA sequence that is to beamplified. “Upstream” is used herein to refer to a location 5, to theDNA sequence to be amplified relative to the coding strand. “Reverseprimers” are primers that contain a region of nucleotides that aresubstantially complementary to a double-stranded DNA template that aredownstream of the DNA sequence that is to be amplified. “Downstream” isused herein to refer to a location 3 to the DNA sequence to be amplifiedrelative to the coding strand.

Any DNA polymerase useful for PCR can be used in the methods disclosedherein. The reagents and polymerase are commercially available from anumber of sources.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. The RNA preferably has 5 □and 3□UTRs. In one embodiment, the 5 □UTR is between one and 3000 nucleotidesin length. The length of 5 □and 3 □UTR sequences to be added to thecoding region can be altered by different methods, including, but notlimited to, designing primers for PCR that anneal to different regionsof the UTRs. Using this approach, one of ordinary skill in the art canmodify the 5 □and 3 □UTR lengths required to achieve optimal translationefficiency following transfection of the transcribed RNA.

The 5 □and 3 □UTRs can be the naturally occurring, endogenous 5 □and 3□UTRs for the nucleic acid of interest. Alternatively, UTR sequencesthat are not endogenous to the nucleic acid of interest can be added byincorporating the UTR sequences into the forward and reverse primers orby any other modifications of the template. The use of UTR sequencesthat are not endogenous to the nucleic acid of interest can be usefulfor modifying the stability and/or translation efficiency of the RNA.For example, it is known that AU-rich elements in 3 □UTR sequences candecrease the stability of mRNA. Therefore, 3 □UTRs can be selected ordesigned to increase the stability of the transcribed RNA based onproperties of UTRs that are well known in the art.

In one embodiment, the 5 □UTR can contain the Kozak sequence of theendogenous nucleic acid. Alternatively, when a 5 □UTR that is notendogenous to the nucleic acid of interest is being added by PCR asdescribed above, a consensus Kozak sequence can be redesigned by addingthe 5 □UTR sequence. Kozak sequences can increase the efficiency oftranslation of some RNA transcripts, but does not appear to be requiredfor all RNAs to enable efficient translation. The requirement for Kozaksequences for many mRNAs is known in the art. In other embodiments the5′ UTR can be 5′UTR of an RNA virus whose RNA genome is stable in cells.In other embodiments various nucleotide analogues can be used in the 3□or 5 □UTR to impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5□end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one preferred embodiment, the promoter isa T7 polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In a preferred embodiment, the mRNA has both a cap on the 5 □end and a 3□poly(A) tail which determine ribosome binding, initiation oftranslation and stability mRNA in the cell. On a circular DNA template,for instance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3 □UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3 □endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3□stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (SEQ ID NO: 31) (size can be 50-5000 T (SEQ ID NO: 32)), orafter PCR by any other method, including, but not limited to, DNAligation or in vitro recombination. Poly(A) tails also provide stabilityto RNAs and reduce their degradation. Generally, the length of a poly(A)tail positively correlates with the stability of the transcribed RNA. Inone embodiment, the poly(A) tail is between 100 and 5000 adenosines (SEQID NO: 33).

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotides(SEQ ID NO: 34) results in about a two-fold increase in the translationefficiency of the RNA. Additionally, the attachment of differentchemical groups to the 3 □end can increase mRNA stability. Suchattachment can contain modified/artificial nucleotides, aptamers andother compounds. For example, ATP analogs can be incorporated into thepoly(A) tail using poly(A) polymerase. ATP analogs can further increasethe stability of the RNA.

5 □caps on also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5□cap. The 5 □cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

RNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation (Amaxa Nucleofector-II(Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (HarvardInstruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver,Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposomemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Non-Viral Delivery Methods

In some aspects, non-viral methods can be used to deliver a nucleic acidencoding a CAR described herein into a cell or tissue or a subject.

In some embodiments, the non-viral method includes the use of atransposon (also called a transposable element). In some embodiments, atransposon is a piece of DNA that can insert itself at a location in agenome, for example, a piece of DNA that is capable of self-replicatingand inserting its copy into a genome, or a piece of DNA that can bespliced out of a longer nucleic acid and inserted into another place ina genome. For example, a transposon comprises a DNA sequence made up ofinverted repeats flanking genes for transposition.

Exemplary methods of nucleic acid delivery using a transposon include aSleeping Beauty transposon system (SBTS) and a piggyBac (PB) transposonsystem. See, e.g., Aronovich et al. Hum. Mol. Genet. 20.R1(2011):R14-20;Singh et al. Cancer Res. 15(2008):2961-2971; Huang et al. Mol. Ther.16(2008):580-589; Grabundzija et al. Mol. Ther. 18(2010):1200-1209;Kebriaei et al. Blood. 122.21(2013):166; Williams. Molecular Therapy16.9(2008):1515-16; Bell et al. Nat. Protoc. 2.12(2007):3153-65; andDing et al. Cell. 122.3(2005):473-83, all of which are incorporatedherein by reference.

The SBTS includes two components: 1) a transposon containing a transgeneand 2) a source of transposase enzyme. The transposase can transpose thetransposon from a carrier plasmid (or other donor DNA) to a target DNA,such as a host cell chromosome/genome. For example, the transposasebinds to the carrier plasmid/donor DNA, cuts the transposon (includingtransgene(s)) out of the plasmid, and inserts it into the genome of thehost cell. See, e.g., Aronovich et al. supra.

Exemplary transposons include a pT2-based transposon. See, e.g.,Grabundzija et al. Nucleic Acids Res. 41.3(2013):1829-47; and Singh etal. Cancer Res. 68.8(2008): 2961-2971, all of which are incorporatedherein by reference. Exemplary transposases include a Tc1/mariner-typetransposase, e.g., the SB10 transposase or the SB11 transposase (ahyperactive transposase which can be expressed, e.g., from acytomegalovirus promoter). See, e.g., Aronovich et al.; Kebriaei et al.;and Grabundzija et al., all of which are incorporated herein byreference.

Use of the SBTS permits efficient integration and expression of atransgene, e.g., a nucleic acid encoding a CAR described herein.Provided herein are methods of generating a cell, e.g., T cell or NKcell, that stably expresses a CAR described herein, e.g., using atransposon system such as SBTS.

In accordance with methods described herein, in some embodiments, one ormore nucleic acids, e.g., plasmids, containing the SBTS components aredelivered to a cell (e.g., T or NK cell). For example, the nucleicacid(s) are delivered by standard methods of nucleic acid (e.g., plasmidDNA) delivery, e.g., methods described herein, e.g., electroporation,transfection, or lipofection. In some embodiments, the nucleic acidcontains a transposon comprising a transgene, e.g., a nucleic acidencoding a CAR described herein. In some embodiments, the nucleic acidcontains a transposon comprising a transgene (e.g., a nucleic acidencoding a CAR described herein) as well as a nucleic acid sequenceencoding a transposase enzyme. In other embodiments, a system with twonucleic acids is provided, e.g., a dual-plasmid system, e.g., where afirst plasmid contains a transposon comprising a transgene, and a secondplasmid contains a nucleic acid sequence encoding a transposase enzyme.For example, the first and the second nucleic acids are co-deliveredinto a host cell.

In some embodiments, cells, e.g., T or NK cells, are generated thatexpress a CAR described herein by using a combination of gene insertionusing the SBTS and genetic editing using a nuclease (e.g., Zinc fingernucleases (ZFNs), Transcription Activator-Like Effector Nucleases(TALENs), the CRISPR/Cas system, or engineered meganucleasere-engineered homing endonucleases).

In some embodiments, use of a non-viral method of delivery permitsreprogramming of cells, e.g., T or NK cells, and direct infusion of thecells into a subject. Advantages of non-viral vectors include but arenot limited to the ease and relatively low cost of producing sufficientamounts required to meet a patient population, stability during storage,and lack of immunogenicity.

Nucleic Acid Constructs Encoding a CAR

The present invention also provides nucleic acid molecules encoding oneor more CAR constructs described herein. In one aspect, the nucleic acidmolecule is provided as a messenger RNA transcript. In one aspect, thenucleic acid molecule is provided as a DNA construct.

Accordingly, in one aspect, the invention pertains to an isolatednucleic acid molecule encoding a chimeric antigen receptor (CAR),wherein the CAR comprises a CD33 binding domain (e.g., a humanized orhuman CD33 binding domain), a transmembrane domain, and an intracellularsignaling domain comprising a stimulatory domain, e.g., a costimulatorysignaling domain and/or a primary signaling domain, e.g., zeta chain. Inone embodiment, the CD33 binding domain is a CD33 binding domaindescribed herein, e.g., an CD33 binding domain which comprises asequence selected from a group consisting of SEQ ID NO:39-47, or asequence with 95-99% identity thereof. In one embodiment, thetransmembrane domain is transmembrane domain of a protein, e.g.,described herein, e.g., selected from the group consisting of the alpha,beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4,CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137and CD154. In one embodiment, the transmembrane domain comprises asequence of SEQ ID NO: 6, or a sequence with 95-99% identity thereof. Inone embodiment, the CD33 binding domain is connected to thetransmembrane domain by a hinge region, e.g., a hinge described herein.In one embodiment, the hinge region comprises SEQ ID NO:2 or SEQ ID NO:3or SEQ ID NO:4 or SEQ ID NO:5, or a sequence with 95-99% identitythereof. In one embodiment, the isolated nucleic acid molecule furthercomprises a sequence encoding a costimulatory domain. In one embodiment,the costimulatory domain is a functional signaling domain of a protein,e.g., described herein, e.g., selected from the group consisting of MHCclass I molecule, TNF receptor proteins, Immunoglobulin-like proteins,cytokine receptors, integrins, signaling lymphocytic activationmolecules (SLAM proteins), activating NK cell receptors, BTLA, a Tollligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD5, ICAM-1,LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CD5, ICAM-1, ICOS (CD278),GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44,NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7Ralpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f,ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX,CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2,TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69,SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligandthat specifically binds with CD83. In one embodiment, the costimulatorydomain comprises a sequence of SEQ ID NO:7, or a sequence with 95-99%identity thereof. In one embodiment, the intracellular signaling domaincomprises a functional signaling domain of 4-1BB and a functionalsignaling domain of CD3 zeta. In one embodiment, the intracellularsignaling domain comprises the sequence of SEQ ID NO: 7 or SEQ ID NO:8,or a sequence with 95-99% identity thereof, and the sequence of SEQ IDNO: 9 or SEQ ID NO:10, or a sequence with 95-99% identity thereof,wherein the sequences comprising the intracellular signaling domain areexpressed in the same frame and as a single polypeptide chain.

In another aspect, the invention pertains to an isolated nucleic acidmolecule encoding a CAR construct comprising a leader sequence of SEQ IDNO: 1, a scFv domain having a sequence selected from the groupconsisting of SEQ ID NOS:39-47, (or a sequence with 95-99% identitythereof), a hinge region of SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 orSEQ ID NO:5 (or a sequence with 95-99% identity thereof), atransmembrane domain having a sequence of SEQ ID NO: 6 (or a sequencewith 95-99% identity thereof), a 4-1BB costimulatory domain having asequence of SEQ ID NO:7 or a CD27 costimulatory domain having a sequenceof SEQ ID NO:8 (or a sequence with 95-99% identity thereof) or a CD28costimulatory domain having a sequence of SEQ ID NO:379 (or a sequencewith 95-99% identity thereof) or a ICOS costimulatory domain having asequence of SEQ ID NO: 381 (or a sequence with 95-99% identity thereof),and a CD3 zeta stimulatory domain having a sequence of SEQ ID NO:9 orSEQ ID NO:10 (or a sequence with 95-99% identity thereof).

In another aspect, the invention pertains to an isolated polypeptidemolecule encoded by the nucleic acid molecule. In one embodiment, theisolated polypeptide molecule comprises a sequence selected from thegroup consisting of SEQ ID NO:48-56, or a sequence with 95-99% identitythereof.

In another aspect, the invention pertains to a nucleic acid moleculeencoding a chimeric antigen receptor (CAR) molecule that comprises aCD33 binding domain, a transmembrane domain, and an intracellularsignaling domain comprising a stimulatory domain, and wherein said CD33binding domain comprises a sequence selected from the group consistingof SEQ ID NO:75-83, or a sequence with 95-99% identity thereof. In oneembodiment, the encoded CAR molecule further comprises a sequenceencoding a costimulatory domain. In one embodiment, the costimulatorydomain is a functional signaling domain of a protein selected from thegroup consisting of MHC class I molecule, TNF receptor proteins,Immunoglobulin-like proteins, cytokine receptors, integrins, signalinglymphocytic activation molecules (SLAM proteins), activating NK cellreceptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28,CD30, CD40, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CD5,ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7,NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D,ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1,ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7,NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1,CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76,PAG/Cbp, CD19a, and a ligand that specifically binds with CD83. In oneembodiment, the costimulatory domain comprises a sequence of SEQ IDNO:7. In one embodiment, the transmembrane domain is a transmembranedomain of a protein selected from the group consisting of the alpha,beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4,CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137,CD154, KIR2DS2, OX40, CD2, CD27, LFA-1 (CD11a and CD18), ICOS (CD278),4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1),NKp44, NKp30, NKp46, CD160, CD19, IL2R (3, IL2R g (Common gamma), IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD,CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c,ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, NKG2D, NKG2C, DNAM1(CD226), SLAMF4, (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108),SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR andPAG/Cbp.

In one embodiment, the transmembrane domain comprises a sequence of SEQID NO:6. In one embodiment, the intracellular signaling domain comprisesa functional signaling domain of 4-1BB and a functional signaling domainof zeta. In one embodiment, the intracellular signaling domain comprisesthe sequence of SEQ ID NO: 7 and the sequence of SEQ ID NO: 9, whereinthe sequences comprising the intracellular signaling domain areexpressed in the same frame and as a single polypeptide chain. In oneembodiment, the CD33 binding domain is connected to the transmembranedomain by a hinge region. In one embodiment, the hinge region comprisesSEQ ID NO:2. In one embodiment, the hinge region comprises SEQ ID NO:3or SEQ ID NO:4 or SEQ ID NO:5.

In another aspect, the invention pertains to an encoded CAR moleculecomprising a leader sequence of SEQ ID NO: 1, a scFv domain having asequence selected from the group consisting of SEQ ID NO:39-47, or asequence with 95-99% identity thereof, a hinge region of SEQ ID NO:2 orSEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5, a transmembrane domain havinga sequence of SEQ ID NO: 6, a 4-1BB costimulatory domain having asequence of SEQ ID NO:7 or a CD27 costimulatory domain having a sequenceof SEQ ID NO:8, and a CD3 zeta stimulatory domain having a sequence ofSEQ ID NO:9 or SEQ ID NO:10. In one embodiment, the encoded CAR moleculecomprises a sequence selected from a group consisting of SEQ IDNO:48-56, or a sequence with 95-99% identity thereof.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity. A retroviral vector may also be, e.g., a gammaretroviralvector. A gammaretroviral vector may include, e.g., a promoter, apackaging signal (w), a primer binding site (PBS), one or more (e.g.,two) long terminal repeats (LTR), and a transgene of interest, e.g., agene encoding a CAR. A gammaretroviral vector may lack viral structuralgens such as gag, pol, and env. Exemplary gammaretroviral vectorsinclude Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV),and Myeloproliferative Sarcoma Virus (MPSV), and vectors derivedtherefrom. Other gammaretroviral vectors are described, e.g., in TobiasMaetzig et al., “Gammaretroviral Vectors: Biology, Technology andApplication” Viruses. 2011 June; 3(6): 677-713.

In another embodiment, the vector comprising the nucleic acid encodingthe desired CAR of the invention is an adenoviral vector (A5/35). Inanother embodiment, the expression of nucleic acids encoding CARs can beaccomplished using of transposons such as sleeping beauty, crisper,CAS9, and zinc finger nucleases. See below June et al. 2009NatureReviews Immunology 9.10: 704-716, is incorporated herein by reference.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The expression constructs of the present invention may also be used fornucleic acid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In another embodiment, theinvention provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al., 2012, MOLECULAR CLONING: ALABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave been shown to contain functional elements downstream of the startsite as well. The spacing between promoter elements frequently isflexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription. Exemplary promoters include theCMV IE gene, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK)promoters.

An example of a promoter that is capable of expressing a CAR transgenein a mammalian T cell is the EF1a promoter. The native EF1a promoterdrives expression of the alpha subunit of the elongation factor-1complex, which is responsible for the enzymatic delivery of aminoacyltRNAs to the ribosome. The EF1a promoter has been extensively used inmammalian expression plasmids and has been shown to be effective indriving CAR expression from transgenes cloned into a lentiviral vector.See, e.g., Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). In oneaspect, the EF1a promoter comprises the sequence provided as SEQ IDNO:11.

Another example of a promoter is the immediate early cytomegalovirus(CMV) promoter sequence. This promoter sequence is a strong constitutivepromoter sequence capable of driving high levels of expression of anypolynucleotide sequence operatively linked thereto. However, otherconstitutive promoter sequences may also be used, including, but notlimited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus (MMTV), human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, as well as human gene promoters such as, but not limitedto, the actin promoter, the myosin promoter, the elongation factor-1αpromoter, the hemoglobin promoter, and the creatine kinase promoter.Further, the invention should not be limited to the use of constitutivepromoters. Inducible promoters are also contemplated as part of theinvention. The use of an inducible promoter provides a molecular switchcapable of turning on expression of the polynucleotide sequence which itis operatively linked when such expression is desired, or turning offthe expression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

Another example of a promoter is the phosphoglycerate kinase (PGK)promoter. In embodiments, a truncated PGK promoter (e.g., a PGK promoterwith one or more, e.g., 1, 2, 5, 10, 100, 200, 300, or 400, nucleotidedeletions when compared to the wild-type PGK promoter sequence) may bedesired. The nucleotide sequences of exemplary PGK promoters areprovided below.

WT PGK Promoter (SEQ ID NO: 384)ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCTGAATCCCCGCCTCGTCCTTCGCAGCGGCCCCCCGGGTGTTCCCATCGCCGCTTCTAGGCCCACTGCGACGCTTGCCTGCACTTCTTACACGCTCTGGGTCCCAGCCGCGGCGACGCAAAGGGCCTTGGTGCGGGTCTCGTCGGCGCAGGGACGCGTTTGGGTCCCGACGGAACCTTTTCCGCGTTGGGGTTGGGG CACCATAAGCTExemplary truncated PGK Promoters: PGK100: (SEQ ID NO: 385)ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGG GTGTGATGGCGGGGTGPGK200: (SEQ ID NO: 386)ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCA GCCGCGCGACGGTAACGPGK300: (SEQ ID NO: 387)ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTT GGAAGGGCTGAATCCCCGPGK400: (SEQ ID NO: 388)ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCTGAATCCCCGCCTCGTCCTTCGCAGCGGCCCCCCGGGTGTTCCCATCGCCGCTTCTAGGCCCACTGCGACGCTTGCCTGCACTTCTTACACGCT CTGGGTCCCAGCCG

A vector may also include, e.g., a signal sequence to facilitatesecretion, a polyadenylation signal and transcription terminator (e.g.,from Bovine Growth Hormone (BGH) gene), an element allowing episomalreplication and replication in prokaryotes (e.g. SV40 origin and ColE1or others known in the art) and/or elements to allow selection (e.g.,ampicillin resistance gene and/or zeocin marker).

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5□flanking region showing the highest level of expression of reportergene is identified as the promoter. Such promoter regions may be linkedto a reporter gene and used to evaluate agents for the ability tomodulate promoter-driven transcription.

In one embodiment, the vector can further comprise a nucleic acidencoding a second CAR. In one embodiment, the second CAR includes anantigen binding domain to a target expressed on acute myeloid leukemiacells, such as, e.g., CD123, CD34, CLL-1, FLT3, or folate receptor beta.In one embodiment, the vector comprises a nucleic acid sequence encodinga first CAR that specifically binds a first antigen and includes anintracellular signaling domain having a costimulatory signaling domainbut not a primary signaling domain, and a nucleic acid encoding a secondCAR that specifically binds a second, different, antigen and includes anintracellular signaling domain having a primary signaling domain but nota costimulatory signaling domain. In one embodiment, the vectorcomprises a nucleic acid encoding a first CD33 CAR that includes a CD33binding domain, a transmembrane domain and a costimulatory domain and anucleic acid encoding a second CAR that specifically binds an antigenother than CD33 (e.g., an antigen expressed on AML cells, e.g., CD123,CD34, CLL-1, FLT3, or folate receptor beta) and includes an antigenbinding domain, a transmembrane domain and a primary signaling domain.In another embodiment, the vector comprises a nucleic acid encoding afirst CD33 CAR that includes a CD33 binding domain, a transmembranedomain and a primary signaling domain and a nucleic acid encoding asecond CAR that specifically binds an antigen other than CD33 (e.g., anantigen expressed on AML cells, e.g., CD123, CLL-1, CD34, FLT3, orfolate receptor beta) and includes an antigen binding domain to theantigen, a transmembrane domain and a costimulatory signaling domain.

In one embodiment, the vector comprises a nucleic acid encoding a CD33CAR described herein and a nucleic acid encoding an inhibitory CAR. Inone embodiment, the inhibitory CAR comprises an antigen binding domainthat binds an antigen found on normal cells but not cancer cells, e.g.,normal cells that also express CD33. In one embodiment, the inhibitoryCAR comprises the antigen binding domain, a transmembrane domain and anintracellular domain of an inhibitory molecule. For example, theintracellular domain of the inhibitory CAR can be an intracellulardomain of PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1,CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4,CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR,A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta.

In embodiments, the vector may comprise two or more nucleic acidsequences encoding a CAR, e.g., a CD33 CAR described herein and a secondCAR, e.g., an inhibitory CAR or a CAR that specifically binds to anantigen other than CD33 (e.g., an antigen expressed on AML cells, e.g.,CD123, CLL-1, CD34, FLT3, or folate receptor beta). In such embodiments,the two or more nucleic acid sequences encoding the CAR are encoded by asingle nucleic molecule in the same frame and as a single polypeptidechain. In this aspect, the two or more CARs, can, e.g., be separated byone or more peptide cleavage sites. (e.g., an auto-cleavage site or asubstrate for an intracellular protease). Examples of peptide cleavagesites include the following, wherein the GSG residues are optional:

T2A: (SEQ ID NO: 389) (GSG)E G R G S L L T C G D V E E N P G P P2A:(SEQ ID NO: 390) (GSG)A T N F S L L K Q A G D V E E N P G P E2A:(SEQ ID NO: 391) (GSG)Q C T N Y A L L K L A G D V E S N P G P F2A:(SEQ ID NO: 392) (GSG)V K Q T L N F D L L K L A G D V E S N P G P

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al., 2012,MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring HarborPress, NY). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle). Other methodsof state-of-the-art targeted delivery of nucleic acids are available,such as delivery of polynucleotides with targeted nanoparticles or othersuitable sub-micron sized delivery system.

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

The present invention further provides a vector comprising a CARencoding nucleic acid molecule. In one aspect, a CAR vector can bedirectly transduced into a cell, e.g., immune effector cell, e.g., a Tcell or NK cell. In one aspect, the vector is a cloning or expressionvector, e.g., a vector including, but not limited to, one or moreplasmids (e.g., expression plasmids, cloning vectors, minicircles,minivectors, double minute chromosomes), retroviral and lentiviralvector constructs. In one aspect, the vector is capable of expressingthe CAR construct in mammalian T cells. In one aspect, the mammalian Tcell is a human T cell.

Sources of Cells

Prior to expansion and genetic modification, a source of cells (e.g.,immune effector cells, e.g., T cells or NK cells) is obtained from asubject. The term “subject” is intended to include living organisms inwhich an immune response can be elicited (e.g., mammals). Examples ofsubjects include humans, dogs, cats, mice, rats, and transgenic speciesthereof. T cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue, cordblood, thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors.

In certain aspects of the present invention, any number of immuneeffector cell (e.g., T cell or NK cell) lines available in the art, maybe used. In certain aspects of the present invention, T cells can beobtained from a unit of blood collected from a subject using any numberof techniques known to the skilled artisan, such as Ficoll™ separation.In one preferred aspect, cells from the circulating blood of anindividual are obtained by apheresis. The apheresis product typicallycontains lymphocytes, including T cells, monocytes, granulocytes, Bcells, other nucleated white blood cells, red blood cells, andplatelets. In one aspect, the cells collected by apheresis may be washedto remove the plasma fraction and to place the cells in an appropriatebuffer or media for subsequent processing steps. In one aspect of theinvention, the cells are washed with phosphate buffered saline (PBS). Inan alternative aspect, the wash solution lacks calcium and may lackmagnesium or may lack many if not all divalent cations.

Initial activation steps in the absence of calcium can lead to magnifiedactivation. As those of ordinary skill in the art would readilyappreciate a washing step may be accomplished by methods known to thosein the art, such as by using a semi-automated “flow-through” centrifuge(for example, the Cobe 2991 cell processor, the Baxter CytoMate, or theHaemonetics Cell Saver 5) according to the manufacturer's instructions.After washing, the cells may be resuspended in a variety ofbiocompatible buffers, such as, for example, Ca-free, Mg-free PBS,PlasmaLyte A, or other saline solution with or without buffer.Alternatively, the undesirable components of the apheresis sample may beremoved and the cells directly resuspended in culture media.

It is recognized that the methods of the application can utilize culturemedia conditions comprising 5% or less, for example 2%, human AB serum,and employ known culture media conditions and compositions, for examplethose described in Smith et al., “Ex vivo expansion of human T cells foradoptive immunotherapy using the novel Xeno-free CTS Immune Cell SerumReplacement” Clinical & Translational Immunology (2015) 4, e31;doi:10.1038/cti.2014.31.

In one aspect, T cells are isolated from peripheral blood lymphocytes bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counterflow centrifugalelutriation.

A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+,CD45RA+, and CD45RO+T cells, can be further isolated by positive ornegative selection techniques. For example, in one aspect, T cells areisolated by incubation with anti-CD3/anti-CD28 (e.g., 3×28)-conjugatedbeads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficientfor positive selection of the desired T cells. In one aspect, the timeperiod is about 30 minutes. In a further aspect, the time period rangesfrom 30 minutes to 36 hours or longer and all integer values therebetween. In a further aspect, the time period is at least 1, 2, 3, 4, 5,or 6 hours. In yet another preferred aspect, the time period is 10 to 24hours. In one aspect, the incubation time period is 24 hours. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmunocompromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainaspects, it may be desirable to perform the selection procedure and usethe “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4+ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain aspects, it may be desirable to enrich foror positively select for regulatory T cells which typically expressCD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certainaspects, T regulatory cells are depleted by anti-C25 conjugated beads orother similar method of selection.

The methods described herein can include, e.g., selection of a specificsubpopulation of immune effector cells, e.g., T cells, that are a Tregulatory cell-depleted population, CD25+ depleted cells, using, e.g.,a negative selection technique, e.g., described herein. Preferably, thepopulation of T regulatory depleted cells contains less than 30%, 25%,20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

In one embodiment, T regulatory cells, e.g., CD25+ T cells, are removedfrom the population using an anti-CD25 antibody, or fragment thereof, ora CD25-binding ligand, IL-2. In one embodiment, the anti-CD25 antibody,or fragment thereof, or CD25-binding ligand is conjugated to asubstrate, e.g., a bead, or is otherwise coated on a substrate, e.g., abead. In one embodiment, the anti-CD25 antibody, or fragment thereof, isconjugated to a substrate as described herein.

In one embodiment, the T regulatory cells, e.g., CD25+ T cells, areremoved from the population using CD25 depletion reagent from Miltenyi™.In one embodiment, the ratio of cells to CD25 depletion reagent is 1e7cells to 20 uL, or 1e7 cells to 15 uL, or 1e7 cells to 10 uL, or 1e7cells to 5 uL, or 1e7 cells to 2.5 uL, or 1e7 cells to 1.25 uL. In oneembodiment, e.g., for T regulatory cells, e.g., CD25+ depletion, greaterthan 500 million cells/ml is used. In a further aspect, a concentrationof cells of 600, 700, 800, or 900 million cells/ml is used.

In one embodiment, the population of immune effector cells to bedepleted includes about 6×10⁹ CD25+ T cells. In other aspects, thepopulation of immune effector cells to be depleted include about 1×10⁹to 1×10¹⁰ CD25+ T cell, and any integer value in between. In oneembodiment, the resulting population T regulatory depleted cells has2×10⁹ T regulatory cells, e.g., CD25+ cells, or less (e.g., 1×10⁹,5×10⁸, 1×10⁸, 5×10⁷, 1×10⁷, or less CD25+ cells).

In one embodiment, the T regulatory cells, e.g., CD25+ cells, areremoved from the population using the CliniMAC system with a depletiontubing set, such as, e.g., tubing 162-01. In one embodiment, theCliniMAC system is run on a depletion setting such as, e.g.,DEPLETION2.1.

Without wishing to be bound by a particular theory, decreasing the levelof negative regulators of immune cells (e.g., decreasing the number ofunwanted immune cells, e.g., T_(REG) cells), in a subject prior toapheresis or during manufacturing of a CAR-expressing cell product canreduce the risk of subject relapse. For example, methods of depletingT_(REG) cells are known in the art. Methods of decreasing T_(REG) cellsinclude, but are not limited to, cyclophosphamide, anti-GITR antibody(an anti-GITR antibody described herein), CD25-depletion, andcombinations thereof.

In some embodiments, the manufacturing methods comprise reducing thenumber of (e.g., depleting) T_(REG) cells prior to manufacturing of theCAR-expressing cell. For example, manufacturing methods comprisecontacting the sample, e.g., the apheresis sample, with an anti-GITRantibody and/or an anti-CD25 antibody (or fragment thereof, or aCD25-binding ligand), e.g., to deplete T_(REG) cells prior tomanufacturing of the CAR-expressing cell (e.g., T cell, NK cell)product.

In an embodiment, a subject is pre-treated with one or more therapiesthat reduce T_(REG) cells prior to collection of cells forCAR-expressing cell product manufacturing, thereby reducing the risk ofsubject relapse to CAR-expressing cell treatment. In an embodiment,methods of decreasing T_(REG) cells include, but are not limited to,administration to the subject of one or more of cyclophosphamide,anti-GITR antibody, CD25-depletion, or a combination thereof.Administration of one or more of cyclophosphamide, anti-GITR antibody,CD25-depletion, or a combination thereof, can occur before, during orafter an infusion of the CAR-expressing cell product.

In an embodiment, a subject is pre-treated with cyclophosphamide priorto collection of cells for CAR-expressing cell product manufacturing,thereby reducing the risk of subject relapse to CAR-expressing celltreatment. In an embodiment, a subject is pre-treated with an anti-GITRantibody prior to collection of cells for CAR-expressing cell productmanufacturing, thereby reducing the risk of subject relapse toCAR-expressing cell treatment.

In one embodiment, the population of cells to be removed are neither theregulatory T cells or tumor cells, but cells that otherwise negativelyaffect the expansion and/or function of CART cells, e.g. cellsexpressing CD14, CD11b, CD33, CD15, or other markers expressed bypotentially immune suppressive cells. In one embodiment, such cells areenvisioned to be removed concurrently with regulatory T cells and/ortumor cells, or following said depletion, or in another order.

The methods described herein can include more than one selection step,e.g., more than one depletion step. Enrichment of a T cell population bynegative selection can be accomplished, e.g., with a combination ofantibodies directed to surface markers unique to the negatively selectedcells. One method is cell sorting and/or selection via negative magneticimmunoadherence or flow cytometry that uses a cocktail of monoclonalantibodies directed to cell surface markers present on the cellsnegatively selected. For example, to enrich for CD4+ cells by negativeselection, a monoclonal antibody cocktail can include antibodies toCD14, CD20, CD11b, CD16, HLA-DR, and CD8.

The methods described herein can further include removing cells from thepopulation which express a tumor antigen, e.g., a tumor antigen thatdoes not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14 orCD11b, to thereby provide a population of T regulatory depleted, e.g.,CD25+ depleted, and tumor antigen depleted cells that are suitable forexpression of a CAR, e.g., a CAR described herein. In one embodiment,tumor antigen expressing cells are removed simultaneously with the Tregulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody, orfragment thereof, and an anti-tumor antigen antibody, or fragmentthereof, can be attached to the same substrate, e.g., bead, which can beused to remove the cells or an anti-CD25 antibody, or fragment thereof,or the anti-tumor antigen antibody, or fragment thereof, can be attachedto separate beads, a mixture of which can be used to remove the cells.In other embodiments, the removal of T regulatory cells, e.g., CD25+cells, and the removal of the tumor antigen expressing cells issequential, and can occur, e.g., in either order.

Also provided are methods that include removing cells from thepopulation which express a check point inhibitor, e.g., a check pointinhibitor described herein, e.g., one or more of PD1+ cells, LAG3+cells, and TIM3+ cells, to thereby provide a population of T regulatorydepleted, e.g., CD25+ depleted cells, and check point inhibitor depletedcells, e.g., PD1+, LAG3+ and/or TIM3+ depleted cells. Exemplary checkpoint inhibitors include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g.,CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 orCD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFRbeta. In one embodiment, check point inhibitor expressing cells areremoved simultaneously with the T regulatory, e.g., CD25+ cells. Forexample, an anti-CD25 antibody, or fragment thereof, and an anti-checkpoint inhibitor antibody, or fragment thereof, can be attached to thesame bead which can be used to remove the cells, or an anti-CD25antibody, or fragment thereof, and the anti-check point inhibitorantibody, or fragment there, can be attached to separate beads, amixture of which can be used to remove the cells. In other embodiments,the removal of T regulatory cells, e.g., CD25+ cells, and the removal ofthe check point inhibitor expressing cells is sequential, and can occur,e.g., in either order.

In one embodiment, a T cell population can be selected that expressesone or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10,IL-13, granzyme B, and perforin, or other appropriate molecules, e.g.,other cytokines. Methods for screening for cell expression can bedetermined, e.g., by the methods described in PCT Publication No.: WO2013/126712.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain aspects, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (e.g., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one aspect, a concentrationof 2 billion cells/ml is used. In one aspect, a concentration of 1billion cells/ml is used. In a further aspect, greater than 100 millioncells/ml is used. In a further aspect, a concentration of cells of 10,15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet oneaspect, a concentration of cells from 75, 80, 85, 90, 95, or 100 millioncells/ml is used. In further aspects, concentrations of 125 or 150million cells/ml can be used.

Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (e.g., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8+ T cells that normally haveweaker CD28 expression.

In a related aspect, it may be desirable to use lower concentrations ofcells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4+ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8+ T cells in dilute concentrations. In one aspect, the concentrationof cells used is 5×10e6/ml. In other aspects, the concentration used canbe from about 1×10⁵/ml to 1×10⁶/ml, and any integer value in between.

In other aspects, the cells may be incubated on a rotator for varyinglengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain aspects, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as immune effector cells, e.g.,T cells or NK cells, isolated and frozen for later use in cell therapy,e.g., T cell therapy, for any number of diseases or conditions thatwould benefit from cell therapy, e.g., T cell therapy, such as thosedescribed herein. In one aspect a blood sample or an apheresis is takenfrom a generally healthy subject. In certain aspects, a blood sample oran apheresis is taken from a generally healthy subject who is at risk ofdeveloping a disease, but who has not yet developed a disease, and thecells of interest are isolated and frozen for later use. In certainaspects, the immune effector cells, e.g., T cells or NK cells may beexpanded, frozen, and used at a later time. In certain aspects, samplesare collected from a patient shortly after diagnosis of a particulardisease as described herein but prior to any treatments. In a furtheraspect, the cells are isolated from a blood sample or an apheresis froma subject prior to any number of relevant treatment modalities,including but not limited to treatment with agents such as natalizumab,efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressiveagents, such as cyclosporin, azathioprine, methotrexate, mycophenolate,and FK506, antibodies, or other immunoablative agents such as CAMPATH,anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506,rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.

In a further aspect of the present invention, T cells are obtained froma patient directly following treatment that leaves the subject withfunctional T cells. In this regard, it has been observed that followingcertain cancer treatments, in particular treatments with drugs thatdamage the immune system, shortly after treatment during the period whenpatients would normally be recovering from the treatment, the quality ofT cells obtained may be optimal or improved for their ability to expandex vivo. Likewise, following ex vivo manipulation using the methodsdescribed herein, these cells may be in a preferred state for enhancedengraftment and in vivo expansion. Thus, it is contemplated within thecontext of the present invention to collect blood cells, including Tcells, dendritic cells, or other cells of the hematopoietic lineage,during this recovery phase. Further, in certain aspects, mobilization(for example, mobilization with GM-CSF) and conditioning regimens can beused to create a condition in a subject wherein repopulation,recirculation, regeneration, and/or expansion of particular cell typesis favored, especially during a defined window of time followingtherapy. Illustrative cell types include T cells, B cells, dendriticcells, and other cells of the immune system.

In one embodiment, the immune effector cells expressing a CAR molecule,e.g., a CAR molecule described herein, are obtained from a subject thathas received a low, immune enhancing dose of an mTOR inhibitor. In anembodiment, the population of immune effector cells, e.g., T cells, tobe engineered to express a CAR, are harvested after a sufficient time,or after sufficient dosing of the low, immune enhancing, dose of an mTORinhibitor, such that the level of PD1 negative immune effector cells,e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g.,T cells/PD1 positive immune effector cells, e.g., T cells, in thesubject or harvested from the subject has been, at least transiently,increased.

In other embodiments, population of immune effector cells, e.g., Tcells, which have, or will be engineered to express a CAR, can betreated ex vivo by contact with an amount of an mTOR inhibitor thatincreases the number of PD1 negative immune effector cells, e.g., Tcells or increases the ratio of PD1 negative immune effector cells,e.g., T cells/PD1 positive immune effector cells, e.g., T cells.

In one embodiment, a T cell population is diaglycerol kinase(DGK)-deficient. DGK-deficient cells include cells that do not expressDGK RNA or protein, or have reduced or inhibited DGK activity.DGK-deficient cells can be generated by genetic approaches, e.g.,administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, toreduce or prevent DGK expression. Alternatively, DGK-deficient cells canbe generated by treatment with DGK inhibitors described herein.

In one embodiment, a T cell population is Ikaros-deficient.Ikaros-deficient cells include cells that do not express Ikaros RNA orprotein, or have reduced or inhibited Ikaros activity, Ikaros-deficientcells can be generated by genetic approaches, e.g., administeringRNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or preventIkaros expression. Alternatively, Ikaros-deficient cells can begenerated by treatment with Ikaros inhibitors, e.g., lenalidomide.

In embodiments, a T cell population is DGK-deficient andIkaros-deficient, e.g., does not express DGK and Ikaros, or has reducedor inhibited DGK and Ikaros activity. Such DGK and Ikaros-deficientcells can be generated by any of the methods described herein.

In an embodiment, the NK cells are obtained from the subject. In anotherembodiment, the NK cells are an NK cell line, e.g., NK-92 cell line(Conkwest).

Allogeneic CAR

In embodiments described herein, the immune effector cell can be anallogeneic immune effector cell, e.g., T cell or NK cell. For example,the cell can be an allogeneic T cell, e.g., an allogeneic T cell lackingexpression of a functional T cell receptor (TCR) and/or human leukocyteantigen (HLA), e.g., HLA class I and/or HLA class II.

A T cell lacking a functional TCR can be, e.g., engineered such that itdoes not express any functional TCR on its surface, engineered such thatit does not express one or more subunits that comprise a functional TCR(e.g., engineered such that it does not express (or exhibits reducedexpression) of TCR alpha, TCR beta, TCR gamma, TCR delta, TCR epsilon,and/or TCR zeta) or engineered such that it produces very littlefunctional TCR on its surface. Alternatively, the T cell can express asubstantially impaired TCR, e.g., by expression of mutated or truncatedforms of one or more of the subunits of the TCR. The term “substantiallyimpaired TCR” means that this TCR will not elicit an adverse immunereaction in a host.

A T cell described herein can be, e.g., engineered such that it does notexpress a functional HLA on its surface. For example, a T cell describedherein, can be engineered such that cell surface expression HLA, e.g.,HLA class 1 and/or HLA class II, is downregulated. In some aspects,downregulation of HLA may be accomplished by reducing or eliminatingexpression of beta-2 microglobulin (B2M).

In some embodiments, the T cell can lack a functional TCR and afunctional HLA, e.g., HLA class I and/or HLA class II.

Modified T cells that lack expression of a functional TCR and/or HLA canbe obtained by any suitable means, including a knock out or knock downof one or more subunit of TCR or HLA. For example, the T cell caninclude a knock down of TCR and/or HLA using siRNA, shRNA, clusteredregularly interspaced short palindromic repeats (CRISPR)transcription-activator like effector nuclease (TALEN), or zinc fingerendonuclease (ZFN).

In some embodiments, the allogeneic cell can be a cell which does notexpresses or expresses at low levels an inhibitory molecule, e.g. a cellengineered by any method described herein. For example, the cell can bea cell that does not express or expresses at low levels an inhibitorymolecule, e.g., that can decrease the ability of a CAR-expressing cellto mount an immune effector response. Examples of inhibitory moleculesinclude PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80,CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR,MHC class I, MHC class II, GAL9, adenosine, and TGFR beta. Inhibition ofan inhibitory molecule, e.g., by inhibition at the DNA, RNA or proteinlevel, can optimize a CAR-expressing cell performance. In embodiments,an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., adsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced shortpalindromic repeats (CRISPR), a transcription-activator like effectornuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., asdescribed herein, can be used.

siRNA and shRNA to Inhibit TCR or HLA

In some embodiments, TCR expression and/or HLA expression can beinhibited using siRNA or shRNA that targets a nucleic acid encoding aTCR and/or HLA, and/or an inhibitory molecule described herein (e.g.,PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, GAL9, adenosine, and TGFR beta), in a T cell.

Expression of siRNA and shRNAs in T cells can be achieved using anyconventional expression system, e.g., such as a lentiviral expressionsystem.

Exemplary shRNAs that downregulate expression of components of the TCRare described, e.g., in US Publication No.: 2012/0321667. ExemplarysiRNA and shRNA that downregulate expression of HLA class I and/or HLAclass II genes are described, e.g., in U.S. publication No.: US2007/0036773.

CRISPR to Inhibit TCR or HLA

“CRISPR” or “CRISPR to TCR and/or HLA” or “CRISPR to inhibit TCR and/orHLA” as used herein refers to a set of clustered regularly interspacedshort palindromic repeats, or a system comprising such a set of repeats.“Cas”, as used herein, refers to a CRISPR-associated protein. A“CRISPR/Cas” system refers to a system derived from CRISPR and Cas whichcan be used to silence or mutate a TCR and/or HLA gene, and/or aninhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4,TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA,BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II,GAL9, adenosine, and TGFR beta).

Naturally-occurring CRISPR/Cas systems are found in approximately 40% ofsequenced eubacteria genomes and 90% of sequenced archaea. Grissa et al.(2007) BMC Bioinformatics 8: 172. This system is a type of prokaryoticimmune system that confers resistance to foreign genetic elements suchas plasmids and phages and provides a form of acquired immunity.Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008)Science 322: 1843-1845.

The CRISPR/Cas system has been modified for use in gene editing(silencing, enhancing or changing specific genes) in eukaryotes such asmice or primates. Wiedenheft et al. (2012) Nature 482: 331-8. This isaccomplished by introducing into the eukaryotic cell a plasmidcontaining a specifically designed CRISPR and one or more appropriateCas.

The CRISPR sequence, sometimes called a CRISPR locus, comprisesalternating repeats and spacers. In a naturally-occurring CRISPR, thespacers usually comprise sequences foreign to the bacterium such as aplasmid or phage sequence; in the TCR and/or HLA CRISPR/Cas system, thespacers are derived from the TCR or HLA gene sequence.

RNA from the CRISPR locus is constitutively expressed and processed byCas proteins into small RNAs. These comprise a spacer flanked by arepeat sequence. The RNAs guide other Cas proteins to silence exogenousgenetic elements at the RNA or DNA level. Horvath et al. (2010) Science327: 167-170; Makarova et al. (2006) Biology Direct 1: 7. The spacersthus serve as templates for RNA molecules, analogously to siRNAs.Pennisi (2013) Science 341: 833-836.

As these naturally occur in many different types of bacteria, the exactarrangements of the CRISPR and structure, function and number of Casgenes and their product differ somewhat from species to species. Haft etal. (2005) PLoS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol.8: R61; Mojica et al. (2005) J Mol. Evol. 60: 174-182; Bolotin et al.(2005) Microbiol. 151: 2551-2561; Pourcel et al. (2005) Microbiol. 151:653-663; and Stern et al. (2010) Trends. Genet. 28: 335-340. Forexample, the Cse (Cas subtype, E. coli) proteins (e.g., CasA) form afunctional complex, Cascade, that processes CRISPR RNA transcripts intospacer-repeat units that Cascade retains. Brouns et al. (2008) Science321: 960-964. In other prokaryotes, Cas6 processes the CRISPRtranscript. The CRISPR-based phage inactivation in E. coli requiresCascade and Cas3, but not Cas1 or Cas2. The Cmr (Cas RAMP module)proteins in Pyrococcus furiosus and other prokaryotes form a functionalcomplex with small CRISPR RNAs that recognizes and cleaves complementarytarget RNAs. A simpler CRISPR system relies on the protein Cas9, whichis a nuclease with two active cutting sites, one for each strand of thedouble helix. Combining Cas9 and modified CRISPR locus RNA can be usedin a system for gene editing. Pennisi (2013) Science 341: 833-836.

The CRISPR/Cas system can thus be used to edit a TCR and/or HLA gene(adding or deleting a basepair), or introducing a premature stop whichthus decreases expression of a TCR and/or HLA. The CRISPR/Cas system canalternatively be used like RNA interference, turning off TCR and/or HLAgene in a reversible fashion. In a mammalian cell, for example, the RNAcan guide the Cas protein to a TCR and/or HLA promoter, stericallyblocking RNA polymerases.

Artificial CRISPR/Cas systems can be generated which inhibit TCR and/orHLA, using technology known in the art, e.g., that described in U.S.Publication No. 20140068797, and Cong (2013) Science 339: 819-823. Otherartificial CRISPR/Cas systems that are known in the art may also begenerated which inhibit TCR and/or HLA, e.g., that described in Tsai(2014) Nature Biotechnol., 32:6 569-576, U.S. Pat. Nos. 8,871,445;8,865,406; 8,795,965; 8,771,945; and 8,697,359.

TALEN to Inhibit TCR and/or HLA

“TALEN” or “TALEN to HLA and/or TCR” or “TALEN to inhibit HLA and/orTCR” refers to a transcription activator-like effector nuclease, anartificial nuclease which can be used to edit the HLA and/or TCR gene,and/or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2,CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3,VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II,GALS, adenosine, and TGFR beta).

TALENs are produced artificially by fusing a TAL effector DNA bindingdomain to a DNA cleavage domain. Transcription activator-like effects(TALEs) can be engineered to bind any desired DNA sequence, including aportion of the HLA or TCR gene. By combining an engineered TALE with aDNA cleavage domain, a restriction enzyme can be produced which isspecific to any desired DNA sequence, including a HLA or TCR sequence.These can then be introduced into a cell, wherein they can be used forgenome editing. Boch (2011) Nature Biotech. 29: 135-6; and Boch et al.(2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501.

TALEs are proteins secreted by Xanthomonas bacteria. The DNA bindingdomain contains a repeated, highly conserved 33-34 amino acid sequence,with the exception of the 12th and 13th amino acids. These two positionsare highly variable, showing a strong correlation with specificnucleotide recognition. They can thus be engineered to bind to a desiredDNA sequence.

To produce a TALEN, a TALE protein is fused to a nuclease (N), which isa wild-type or mutated FokI endonuclease. Several mutations to FokI havebeen made for its use in TALENs; these, for example, improve cleavagespecificity or activity. Cermak et al. (2011) Nucl. Acids Res. 39: e82;Miller et al. (2011) Nature Biotech. 29: 143-8; Hockemeyer et al. (2011)Nature Biotech. 29: 731-734; Wood et al. (2011) Science 333: 307; Doyonet al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) NatureBiotech. 25: 786-793; and Guo et al. (2010) J Mol. Biol. 200: 96.

The FokI domain functions as a dimer, requiring two constructs withunique DNA binding domains for sites in the target genome with properorientation and spacing. Both the number of amino acid residues betweenthe TALE DNA binding domain and the FokI cleavage domain and the numberof bases between the two individual TALEN binding sites appear to beimportant parameters for achieving high levels of activity. Miller etal. (2011) Nature Biotech. 29: 143-8.

A HLA or TCR TALEN can be used inside a cell to produce adouble-stranded break (DSB). A mutation can be introduced at the breaksite if the repair mechanisms improperly repair the break vianon-homologous end joining. For example, improper repair may introduce aframe shift mutation. Alternatively, foreign DNA can be introduced intothe cell along with the TALEN; depending on the sequences of the foreignDNA and chromosomal sequence, this process can be used to correct adefect in the HLA or TCR gene or introduce such a defect into a wt HLAor TCR gene, thus decreasing expression of HLA or TCR.

TALENs specific to sequences in HLA or TCR can be constructed using anymethod known in the art, including various schemes using modularcomponents. Zhang et al. (2011) Nature Biotech. 29: 149-53; Geibler etal. (2011) PLoS ONE 6: e19509.

Zinc Finger Nuclease to Inhibit HLA and/or TCR

“ZFN” or “Zinc Finger Nuclease” or “ZFN to HLA and/or TCR” or “ZFN toinhibit HLA and/or TCR” refer to a zinc finger nuclease, an artificialnuclease which can be used to edit the HLA and/or TCR gene, and/or aninhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4,TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA,BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MEW class II,GAL9, adenosine, and TGFR beta).

Like a TALEN, a ZFN comprises a FokI nuclease domain (or derivativethereof) fused to a DNA-binding domain. In the case of a ZFN, theDNA-binding domain comprises one or more zinc fingers. Carroll et al.(2011) Genetics Society of America 188: 773-782; and Kim et al. (1996)Proc. Natl. Acad. Sci. USA 93: 1156-1160.

A zinc finger is a small protein structural motif stabilized by one ormore zinc ions. A zinc finger can comprise, for example, Cys2His2, andcan recognize an approximately 3-bp sequence. Various zinc fingers ofknown specificity can be combined to produce multi-finger polypeptideswhich recognize about 6, 9, 12, 15 or 18-bp sequences. Various selectionand modular assembly techniques are available to generate zinc fingers(and combinations thereof) recognizing specific sequences, includingphage display, yeast one-hybrid systems, bacterial one-hybrid andtwo-hybrid systems, and mammalian cells.

Like a TALEN, a ZFN must dimerize to cleave DNA. Thus, a pair of ZFNsare required to target non-palindromic DNA sites. The two individualZFNs must bind opposite strands of the DNA with their nucleases properlyspaced apart. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95:10570-5.

Also like a TALEN, a ZFN can create a double-stranded break in the DNA,which can create a frame-shift mutation if improperly repaired, leadingto a decrease in the expression and amount of HLA and/or TCR in a cell.ZFNs can also be used with homologous recombination to mutate in the HLAor TCR gene.

ZFNs specific to sequences in HLA AND/OR TCR can be constructed usingany method known in the art. See, e.g., Provasi (2011) Nature Med. 18:807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008)Mol. Ther. 16: 1200-7; Guo et al. (2010) J Mol. Biol. 400: 96; U.S.Patent Publication 2011/0158957; and U.S. Patent Publication2012/0060230.

Telomerase Expression

While not wishing to be bound by any particular theory, in someembodiments, a therapeutic T cell has short term persistence in apatient, due to shortened telomeres in the T cell; accordingly,transfection with a telomerase gene can lengthen the telomeres of the Tcell and improve persistence of the T cell in the patient. See CarlJune, “Adoptive T cell therapy for cancer in the clinic”, Journal ofClinical Investigation, 117:1466-1476 (2007). Thus, in an embodiment, animmune effector cell, e.g., a T cell, ectopically expresses a telomerasesubunit, e.g., the catalytic subunit of telomerase, e.g., TERT, e.g.,hTERT. In some aspects, this disclosure provides a method of producing aCAR-expressing cell, comprising contacting a cell with a nucleic acidencoding a telomerase subunit, e.g., the catalytic subunit oftelomerase, e.g., TERT, e.g., hTERT. The cell may be contacted with thenucleic acid before, simultaneous with, or after being contacted with aconstruct encoding a CAR.

In one aspect, the disclosure features a method of making a populationof immune effector cells (e.g., T cells, NK cells). In an embodiment,the method comprises: providing a population of immune effector cells(e.g., T cells or NK cells), contacting the population of immuneeffector cells with a nucleic acid encoding a CAR; and contacting thepopulation of immune effector cells with a nucleic acid encoding atelomerase subunit, e.g., hTERT, under conditions that allow for CAR andtelomerase expression.

In an embodiment, the nucleic acid encoding the telomerase subunit isDNA. In an embodiment, the nucleic acid encoding the telomerase subunitcomprises a promoter capable of driving expression of the telomerasesubunit.

In an embodiment, hTERT has the amino acid sequence of GenBank ProteinID AAC51724.1 (Meyerson et al., “hEST2, the Putative Human TelomeraseCatalytic Subunit Gene, Is Up-Regulated in Tumor Cells and duringImmortalization” Cell Volume 90, Issue 4, 22 Aug. 1997, Pages 785-795)as follows:

(SEQ ID NO: 157) MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRGCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPA LPSDFKTILD

In an embodiment, the hTERT has a sequence at least 80%, 85%, 90%, 95%,96{circumflex over ( )}, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 157. In an embodiment, the hTERT has a sequence of SEQ ID NO:157. In an embodiment, the hTERT comprises a deletion (e.g., of no morethan 5, 10, 15, 20, or 30 amino acids) at the N-terminus, theC-terminus, or both. In an embodiment, the hTERT comprises a transgenicamino acid sequence (e.g., of no more than 5, 10, 15, 20, or 30 aminoacids) at the N-terminus, the C-terminus, or both.

In an embodiment, the hTERT is encoded by the nucleic acid sequence ofGenBank Accession No. AF018167 (Meyerson et al., “hEST2, the PutativeHuman Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cellsand during Immortalization” Cell Volume 90, Issue 4, 22 Aug. 1997, Pages785-795):

(SEQ ID NO: 158) 1 caggcagcgt ggtcctgctg cgcacgtggg aagccctggccccggccacc cccgcgatgc 61 cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcgcagccactac cgcgaggtgc 121 tgccgctggc cacgttcgtg cggcgcctgg ggccccagggctggcggctg gtgcagcgcg 181 gggacccggc ggctttccgc gcgctggtgg cccagtgcctggtgtgcgtg ccctgggacg 241 cacggccgcc ccccgccgcc ccctccttcc gccaggtgtcctgcctgaag gagctggtgg 301 cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaacgtgctggcc ttcggcttcg 361 cgctgctgga cggggcccgc gggggccccc ccgaggccttcaccaccagc gtgcgcagct 421 acctgcccaa cacggtgacc gacgcactgc gggggagcggggcgtggggg ctgctgttgc 481 gccgcgtggg cgacgacgtg ctggttcacc tgctggcacgctgcgcgctc tttgtgctgg 541 tggctcccag ctgcgcctac caggtgtgcg ggccgccgctgtaccagctc ggcgctgcca 601 ctcaggcccg gcccccgcca cacgctagtg gaccccgaaggcgtctggga tgcgaacggg 661 cctggaacca tagcgtcagg gaggccgggg tccccctgggcctgccagcc ccgggtgcga 721 ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcccaagaggccc aggcgtggcg 781 ctgcccctga gccggagcgg acgcccgttg ggcaggggtcctgggcccac ccgggcagga 841 cgcgtggacc gagtgaccgt ggtttctgtg tggtgtcacctgccagaccc gccgaagaag 901 ccacctcttt ggagggtgcg ctctctggca cgcgccactcccacccatcc gtgggccgcc 961 agcaccacgc gggcccccca tccacatcgc ggccaccacgtccctgggac acgccttgtc 1021 ccccggtgta cgccgagacc aagcacttcc tctactcctcaggcgacaag gagcagctgc 1081 ggccctcctt cctactcagc tctctgaggc ccagcctgactggcgctcgg aggctcgtgg 1141 agaccatctt tctgggttcc aggccctgga tgccagggactccccgcagg ttgccccgcc 1201 tgccccagcg ctactggcaa atgcggcccc tgtttctggagctgcttggg aaccacgcgc 1261 agtgccccta cggggtgctc ctcaagacgc actgcccgctgcgagctgcg gtcaccccag 1321 cagccggtgt ctgtgcccgg gagaagcccc agggctctgtggcggccccc gaggaggagg 1381 acacagaccc ccgtcgcctg gtgcagctgc tccgccagcacagcagcccc tggcaggtgt 1441 acggcttcgt gcgggcctgc ctgcgccggc tggtgcccccaggcctctgg ggctccaggc 1501 acaacgaacg ccgcttcctc aggaacacca agaagttcatctccctgggg aagcatgcca 1561 agctctcgct gcaggagctg acgtggaaga tgagcgtgcggggctgcgct tggctgcgca 1621 ggagcccagg ggttggctgt gttccggccg cagagcaccgtctgcgtgag gagatcctgg 1681 ccaagttcct gcactggctg atgagtgtgt acgtcgtcgagctgctcagg tctttctttt 1741 atgtcacgga gaccacgttt caaaagaaca ggctctttttctaccggaag agtgtctgga 1801 gcaagttgca aagcattgga atcagacagc acttgaagagggtgcagctg cgggagctgt 1861 cggaagcaga ggtcaggcag catcgggaag ccaggcccgccctgctgacg tccagactcc 1921 gcttcatccc caagcctgac gggctgcggc cgattgtgaacatggactac gtcgtgggag 1981 ccagaacgtt ccgcagagaa aagagggccg agcgtctcacctcgagggtg aaggcactgt 2041 tcagcgtgct caactacgag cgggcgcggc gccccggcctcctgggcgcc tctgtgctgg 2101 gcctggacga tatccacagg gcctggcgca ccttcgtgctgcgtgtgcgg gcccaggacc 2161 cgccgcctga gctgtacttt gtcaaggtgg atgtgacgggcgcgtacgac accatccccc 2221 aggacaggct cacggaggtc atcgccagca tcatcaaaccccagaacacg tactgcgtgc 2281 gtcggtatgc cgtggtccag aaggccgccc atgggcacgtccgcaaggcc ttcaagagcc 2341 acgtctctac cttgacagac ctccagccgt acatgcgacagttcgtggct cacctgcagg 2401 agaccagccc gctgagggat gccgtcgtca tcgagcagagctcctccctg aatgaggcca 2461 gcagtggcct cttcgacgtc ttcctacgct tcatgtgccaccacgccgtg cgcatcaggg 2521 gcaagtccta cgtccagtgc caggggatcc cgcagggctccatcctctcc acgctgctct 2581 gcagcctgtg ctacggcgac atggagaaca agctgtttgcggggattcgg cgggacgggc 2641 tgctcctgcg tttggtggat gatttcttgt tggtgacacctcacctcacc cacgcgaaaa 2701 ccttcctcag gaccctggtc cgaggtgtcc ctgagtatggctgcgtggtg aacttgcgga 2761 agacagtggt gaacttccct gtagaagacg aggccctgggtggcacggct tttgttcaga 2821 tgccggccca cggcctattc ccctggtgcg gcctgctgctggatacccgg accctggagg 2881 tgcagagcga ctactccagc tatgcccgga cctccatcagagccagtctc accttcaacc 2941 gcggcttcaa ggctgggagg aacatgcgtc gcaaactctttggggtcttg cggctgaagt 3001 gtcacagcct gtttctggat ttgcaggtga acagcctccagacggtgtgc accaacatct 3061 acaagatcct cctgctgcag gcgtacaggt ttcacgcatgtgtgctgcag ctcccatttc 3121 atcagcaagt ttggaagaac cccacatttt tcctgcgcgtcatctctgac acggcctccc 3181 tctgctactc catcctgaaa gccaagaacg cagggatgtcgctgggggcc aagggcgccg 3241 ccggccctct gccctccgag gccgtgcagt ggctgtgccaccaagcattc ctgctcaagc 3301 tgactcgaca ccgtgtcacc tacgtgccac tcctggggtcactcaggaca gcccagacgc 3361 agctgagtcg gaagctcccg gggacgacgc tgactgccctggaggccgca gccaacccgg 3421 cactgccctc agacttcaag accatcctgg actgatggccacccgcccac agccaggccg 3481 agagcagaca ccagcagccc tgtcacgccg ggctctacgtcccagggagg gaggggcggc 3541 ccacacccag gcccgcaccg ctgggagtct gaggcctgagtgagtgtttg gccgaggcct 3601 gcatgtccgg ctgaaggctg agtgtccggc tgaggcctgagcgagtgtcc agccaagggc 3661 tgagtgtcca gcacacctgc cgtcttcact tccccacaggctggcgctcg gctccacccc 3721 agggccagct tttcctcacc aggagcccgg cttccactccccacatagga atagtccatc 3781 cccagattcg ccattgttca cccctcgccc tgccctcctttgccttccac ccccaccatc 3841 caggtggaga ccctgagaag gaccctggga gctctgggaatttggagtga ccaaaggtgt 3901 gccctgtaca caggcgagga ccctgcacct ggatgggggtccctgtgggt caaattgggg 3961 ggaggtgctg tgggagtaaa atactgaata tatgagtttttcagttttga aaaaaaaaaa 4021 aaaaaaa

In an embodiment, the hTERT is encoded by a nucleic acid having asequence at least 80%, 85%, 90%, 95%, 96, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 158. In an embodiment, the hTERT is encodedby a nucleic acid of SEQ ID NO: 158.

Activation and Expansion of T Cells

T cells may be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005.

Generally, the T cells of the invention may be expanded by contact witha surface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a costimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and ananti-CD28 antibody can be used. Examples of an anti-CD28 antibodyinclude 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used ascan other methods commonly known in the art (Berg et al., TransplantProc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med.190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63,1999).

In certain aspects, the primary stimulatory signal and the costimulatorysignal for the T cell may be provided by different protocols. Forexample, the agents providing each signal may be in solution or coupledto a surface. When coupled to a surface, the agents may be coupled tothe same surface (i.e., in “cis” formation) or to separate surfaces(i.e., in “trans” formation). Alternatively, one agent may be coupled toa surface and the other agent in solution. In one aspect, the agentproviding the costimulatory signal is bound to a cell surface and theagent providing the primary activation signal is in solution or coupledto a surface. In certain aspects, both agents can be in solution. In oneaspect, the agents may be in soluble form, and then cross-linked to asurface, such as a cell expressing Fc receptors or an antibody or otherbinding agent which will bind to the agents. In this regard, see forexample, U.S. Patent Application Publication Nos. 20040101519 and20060034810 for artificial antigen presenting cells (aAPCs) that arecontemplated for use in activating and expanding T cells in the presentinvention.

In one aspect, the two agents are immobilized on beads, either on thesame bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way ofexample, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the costimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one aspect, a 1:1ratio of each antibody bound to the beads for CD4+ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular aspect an increase offrom about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one aspect, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain aspects of theinvention, the ratio of anti CD28 antibody to anti CD3 antibody bound tothe beads is greater than 2:1. In one particular aspect, a 1:100CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:75CD3:CD28 ratio of antibody bound to beads is used. In a further aspect,a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In one aspect,a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In onepreferred aspect, a 1:10 CD3:CD28 ratio of antibody bound to beads isused. In one aspect, a 1:3 CD3:CD28 ratio of antibody bound to the beadsis used. In yet one aspect, a 3:1 CD3:CD28 ratio of antibody bound tothe beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain aspects the ratio of cells toparticles ranges from 1:100 to 100:1 and any integer values in-betweenand in further aspects the ratio comprises 1:9 to 9:1 and any integervalues in between, can also be used to stimulate T cells. The ratio ofanti-CD3- and anti-CD28-coupled particles to T cells that result in Tcell stimulation can vary as noted above, however certain preferredvalues include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6,1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,and 15:1 with one preferred ratio being at least 1:1 particles per Tcell. In one aspect, a ratio of particles to cells of 1:1 or less isused. In one particular aspect, a preferred particle: cell ratio is 1:5.In further aspects, the ratio of particles to cells can be varieddepending on the day of stimulation. For example, in one aspect, theratio of particles to cells is from 1:1 to 10:1 on the first day andadditional particles are added to the cells every day or every other daythereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (basedon cell counts on the day of addition). In one particular aspect, theratio of particles to cells is 1:1 on the first day of stimulation andadjusted to 1:5 on the third and fifth days of stimulation. In oneaspect, particles are added on a daily or every other day basis to afinal ratio of 1:1 on the first day, and 1:5 on the third and fifth daysof stimulation. In one aspect, the ratio of particles to cells is 2:1 onthe first day of stimulation and adjusted to 1:10 on the third and fifthdays of stimulation. In one aspect, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type. In one aspect, the most typical ratiosfor use are in the neighborhood of 1:1, 2:1 and 3:1 on the first day.

In further aspects of the present invention, the cells, such as T cells,are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative aspect, prior to culture, the agent-coated beads and cellsare not separated but are cultured together. In a further aspect, thebeads and cells are first concentrated by application of a force, suchas a magnetic force, resulting in increased ligation of cell surfacemarkers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one aspect the cells (for example, 10⁴to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer, forexample PBS (without divalent cations such as, calcium and magnesium).Again, those of ordinary skill in the art can readily appreciate anycell concentration may be used. For example, the target cell may be veryrare in the sample and comprise only 0.01% of the sample or the entiresample (i.e., 100%) may comprise the target cell of interest.Accordingly, any cell number is within the context of the presentinvention. In certain aspects, it may be desirable to significantlydecrease the volume in which particles and cells are mixed together(i.e., increase the concentration of cells), to ensure maximum contactof cells and particles. For example, in one aspect, a concentration ofabout 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6billion/ml, 5 billion/ml, or 2 billion cells/ml is used. In one aspect,greater than 100 million cells/ml is used. In a further aspect, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet one aspect, a concentration of cells from 75,80, 85, 90, 95, or 100 million cells/ml is used. In further aspects,concentrations of 125 or 150 million cells/ml can be used. Using highconcentrations can result in increased cell yield, cell activation, andcell expansion. Further, use of high cell concentrations allows moreefficient capture of cells that may weakly express target antigens ofinterest, such as CD28-negative T cells. Such populations of cells mayhave therapeutic value and would be desirable to obtain in certainaspects. For example, using high concentration of cells allows moreefficient selection of CD8+ T cells that normally have weaker CD28expression.

In one embodiment, cells transduced with a nucleic acid encoding a CAR,e.g., a CAR described herein, are expanded, e.g., by a method describedherein. In one embodiment, the cells are expanded in culture for aperiod of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18,21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 days). In one embodiment, the cells are expanded for a periodof 4 to 9 days. In one embodiment, the cells are expanded for a periodof 8 days or less, e.g., 7, 6 or 5 days. In one embodiment, the cells,e.g., a CD33 CAR cell described herein, are expanded in culture for 5days, and the resulting cells are more potent than the same cellsexpanded in culture for 9 days under the same culture conditions.Potency can be defined, e.g., by various T cell functions, e.g.proliferation, target cell killing, cytokine production, activation,migration, or combinations thereof. In one embodiment, the cells, e.g.,a CD33 CAR cell described herein, expanded for 5 days show at least aone, two, three or four fold increase in cells doublings upon antigenstimulation as compared to the same cells expanded in culture for 9 daysunder the same culture conditions. In one embodiment, the cells, e.g.,the cells expressing a CD33 CAR described herein, are expanded inculture for 5 days, and the resulting cells exhibit higherproinflammatory cytokine production, e.g., IFN-γ and/or GM-CSF levels,as compared to the same cells expanded in culture for 9 days under thesame culture conditions. In one embodiment, the cells, e.g., a CD33 CARcell described herein, expanded for 5 days show at least a one, two,three, four, five, ten fold or more increase in pg/ml of proinflammatorycytokine production, e.g., IFN-γ and/or GM-CSF levels, as compared tothe same cells expanded in culture for 9 days under the same cultureconditions.

In one aspect of the present invention, the mixture may be cultured forseveral hours (about 3 hours) to about 14 days or any hourly integervalue in between. In one aspect, the mixture may be cultured for 21days. In one aspect of the invention the beads and the T cells arecultured together for about eight days. In one aspect, the beads and Tcells are cultured together for 2-3 days. Several cycles of stimulationmay also be desired such that culture time of T cells can be 60 days ormore. Conditions appropriate for T cell culture include an appropriatemedia (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15,(Lonza)) that may contain factors necessary for proliferation andviability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

In one embodiment, the cells are expanded in an appropriate media (e.g.,media described herein) that includes one or more interleukin thatresult in at least a 200-fold (e.g., 200-fold, 250-fold, 300-fold,350-fold) increase in cells over a 14 day expansion period, e.g., asmeasured by a method described herein such as flow cytometry. In oneembodiment, the cells are expanded in the presence of IL-15 and/or IL-7(e.g., IL-15 and IL-7).

In embodiments, methods described herein, e.g., CAR-expressing cellmanufacturing methods, comprise removing T regulatory cells, e.g., CD25+T cells, from a cell population, e.g., using an anti-CD25 antibody, orfragment thereof, or a CD25-binding ligand, IL-2. Methods of removing Tregulatory cells, e.g., CD25+ T cells, from a cell population aredescribed herein. In embodiments, the methods, e.g., manufacturingmethods, further comprise contacting a cell population (e.g., a cellpopulation in which T regulatory cells, such as CD25+ T cells, have beendepleted; or a cell population that has previously contacted ananti-CD25 antibody, fragment thereof, or CD25-binding ligand) with IL-15and/or IL-7. For example, the cell population (e.g., that has previouslycontacted an anti-CD25 antibody, fragment thereof, or CD25-bindingligand) is expanded in the presence of IL-15 and/or IL-7.

In some embodiments a CAR-expressing cell described herein is contactedwith a composition comprising a interleukin-15 (IL-15) polypeptide, ainterleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination ofboth a IL-15 polypeptide and a IL-15Ra polypeptide e.g., hetIL-15,during the manufacturing of the CAR-expressing cell, e.g., ex vivo. Inembodiments, a CAR-expressing cell described herein is contacted with acomposition comprising a IL-15 polypeptide during the manufacturing ofthe CAR-expressing cell, e.g., ex vivo. In embodiments, a CAR-expressingcell described herein is contacted with a composition comprising acombination of both a IL-15 polypeptide and a IL-15 Ra polypeptideduring the manufacturing of the CAR-expressing cell, e.g., ex vivo. Inembodiments, a CAR-expressing cell described herein is contacted with acomposition comprising hetIL-15 during the manufacturing of theCAR-expressing cell, e.g., ex vivo.

In one embodiment the CAR-expressing cell described herein is contactedwith a composition comprising hetIL-15 during ex vivo expansion. In anembodiment, the CAR-expressing cell described herein is contacted with acomposition comprising an IL-15 polypeptide during ex vivo expansion. Inan embodiment, the CAR-expressing cell described herein is contactedwith a composition comprising both an IL-15 polypeptide and an IL-15Rapolypeptide during ex vivo expansion. In one embodiment the contactingresults in the survival and proliferation of a lymphocyte subpopulation,e.g., CD8+ T cells.

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (TH, CD4+) that is greater than the cytotoxic or suppressor Tcell population (TC, CD8+). Ex vivo expansion of T cells by stimulatingCD3 and CD28 receptors produces a population of T cells that prior toabout days 8-9 consists predominately of TH cells, while after aboutdays 8-9, the population of T cells comprises an increasingly greaterpopulation of TC cells. Accordingly, depending on the purpose oftreatment, infusing a subject with a T cell population comprisingpredominately of TH cells may be advantageous. Similarly, if anantigen-specific subset of TC cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Once a CD33 CAR is constructed, various assays can be used to evaluatethe activity of the molecule, such as but not limited to, the ability toexpand T cells following antigen stimulation, sustain T cell expansionin the absence of re-stimulation, and anti-cancer activities inappropriate in vitro and animal models. Assays to evaluate the effectsof a CD33 CAR are described in further detail below.

Western blot analysis of CAR expression in primary T cells can be usedto detect the presence of monomers and dimers. See, e.g., Milone et al.,Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, T cells (1:1mixture of CD4⁺ and CD8⁺ T cells) expressing the CARs are expanded invitro for more than 10 days followed by lysis and SDS-PAGE underreducing conditions. CARs containing the full length TCR-t cytoplasmicdomain and the endogenous TCR-ζ chain are detected by western blottingusing an antibody to the TCR-chain. The same T cell subsets are used forSDS-PAGE analysis under non-reducing conditions to permit evaluation ofcovalent dimer formation.

In vitro expansion of CAR⁺ T cells following antigen stimulation can bemeasured by flow cytometry. For example, a mixture of CD4⁺ and CD8⁺ Tcells are stimulated with αCD³/αCD28 aAPCs followed by transduction withlentiviral vectors expressing GFP under the control of the promoters tobe analyzed. Exemplary promoters include the CMV IE gene, EF-1α,ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescenceis evaluated on day 6 of culture in the CD4⁺ and/or CD8⁺ T cell subsetsby flow cytometry. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Alternatively, a mixture of CD4⁺ and CD8⁺ T cells arestimulated with αCD3/αCD28 coated magnetic beads on day 0, andtransduced with CAR on day 1 using a bicistronic lentiviral vectorexpressing CAR along with eGFP using a 2A ribosomal skipping sequence.Cultures are re-stimulated with either CD19⁺ K562 cells (K562-CD19),wild-type K562 cells (K562 wild type) or K562 cells expressing hCD32 and4-1BBL in the presence of antiCD3 and anti-CD28 antibody (K562-BBL-3/28)following washing. Exogenous IL-2 is added to the cultures every otherday at 100 IU/ml. GFP⁺ T cells are enumerated by flow cytometry usingbead-based counting. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Similar assays can be performed using anti-CD123 Tcells (see, e.g. Gill et al Blood 2014; 123:2343) or with anti-CD33 CARTcells.

Sustained CAR⁺ T cell expansion in the absence of re-stimulation canalso be measured. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, mean T cell volume (fl) is measured on day 8of culture using a Coulter Multisizer III particle counter, a NexcelomCellometer Vision or Millipore Scepter, following stimulation withαCD3/αCD28 coated magnetic beads on day 0, and transduction with theindicated CAR on day 1.

Animal models can also be used to measure a CART activity. For example,xenograft model using human CD19-specific CAR⁺ T cells to treat aprimary human pre-B ALL in immunodeficient mice can be used. See, e.g.,Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Very briefly,after establishment of ALL, mice are randomized as to treatment groups.Different numbers of αCD19-ζ and αCD19-BB-ζ engineered T cells arecoinjected at a 1:1 ratio into NOD-SCID-γ^(−/−) mice bearing B-ALL. Thenumber of copies of αCD19-ζ and αCD19-BB-ζ vector in spleen DNA frommice is evaluated at various times following T cell injection. Animalsare assessed for leukemia at weekly intervals. Peripheral bloodCD19⁺B-ALL blast cell counts are measured in mice that are injected withαCD19-ζ CAR⁺ T cells or mock-transduced T cells. Survival curves for thegroups are compared using the log-rank test. In addition, absoluteperipheral blood CD4⁺ and CD8⁺ T cell counts 4 weeks following T cellinjection in NOD-SCID-γ^(−/−) mice can also be analyzed. Mice areinjected with leukemic cells and 3 weeks later are injected with T cellsengineered to express CAR by a bicistronic lentiviral vector thatencodes the CAR linked to eGFP. T cells are normalized to 45-50% inputGFP⁺ T cells by mixing with mock-transduced cells prior to injection,and confirmed by flow cytometry. Animals are assessed for leukemia at1-week intervals. Survival curves for the CAR⁺ T cell groups arecompared using the log-rank test. Similar experiments can be done withCD33 CARTS.

Dose dependent CAR treatment response can be evaluated. See, e.g.,Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). For example,peripheral blood is obtained 35-70 days after establishing leukemia inmice injected on day 21 with CAR T cells, an equivalent number ofmock-transduced T cells, or no T cells. Mice from each group arerandomly bled for determination of peripheral blood CD19⁺ ALL blastcounts and then killed on days 35 and 49. The remaining animals areevaluated on days 57 and 70. Similar experiments can be done with CD33CARTS.

Assessment of cell proliferation and cytokine production has beenpreviously described, e.g., at Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, assessment of CAR-mediated proliferation isperformed in microtiter plates by mixing washed T cells with K562 cellsexpressing CD19 (1(19) or CD32 and CD137 (KT32-BBL) for a finalT-cell:K562 ratio of 2:1. K562 cells are irradiated with gamma-radiationprior to use. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonalantibodies are added to cultures with KT32-BBL cells to serve as apositive control for stimulating T-cell proliferation since thesesignals support long-term CD8⁺ T cell expansion ex vivo. T cells areenumerated in cultures using CountBright™ fluorescent beads (Invitrogen,Carlsbad, Calif.) and flow cytometry as described by the manufacturer.CAR⁺ T cells are identified by GFP expression using T cells that areengineered with eGFP-2A linked CAR-expressing lentiviral vectors. ForCAR+ T cells not expressing GFP, the CAR+ T cells are detected withbiotinylated recombinant CD33 protein and a secondary avidin-PEconjugate. CD4+ and CD8⁺ expression on T cells are also simultaneouslydetected with specific monoclonal antibodies (BD Biosciences). Cytokinemeasurements are performed on supernatants collected 24 hours followingre-stimulation using the human TH1/TH2 cytokine cytometric bead arraykit (BD Biosciences, San Diego, Calif.) according the manufacturer'sinstructions or using a Luminex 30-plex kit (Invitrogen). Fluorescenceis assessed using a BD Fortessa flow cytometer, and data is analyzedaccording to the manufacturer's instructions. Similar experiments can bedone with CD33 CARTS.

Cytotoxicity can be assessed by a standard 51Cr-release assay. See,e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly,target cells (K562 lines and primary pro-B-ALL cells) are loaded with51Cr (as NaCrO4, New England Nuclear, Boston, Mass.) at 37° C. for 2hours with frequent agitation, washed twice in complete RPMI and platedinto microtiter plates. Effector T cells are mixed with target cells inthe wells in complete RPMI at varying ratios of effector cell:targetcell (E:T). Additional wells containing media only (spontaneous release,SR) or a 1% solution of triton-X 100 detergent (total release, TR) arealso prepared. After 4 hours of incubation at 37° C., supernatant fromeach well is harvested. Released 51Cr is then measured using a gammaparticle counter (Packard Instrument Co., Waltham, Mass.). Eachcondition is performed in at least triplicate, and the percentage oflysis is calculated using the formula: % Lysis=(ER−SR)/(TR−SR), where ERrepresents the average 51Cr released for each experimental condition.

Imaging technologies can be used to evaluate specific trafficking andproliferation of CARs in tumor-bearing animal models. Such assays havebeen described, for example, in Barrett et al., Human Gene Therapy22:1575-1586 (2011). Briefly, NOD/SCID/γc^(−/−) (NSG) mice are injectedIV with Nalm-6 cells followed 7 days later with T cells 4 hour afterelectroporation with the CAR constructs. The T cells are stablytransfected with a lentiviral construct to express firefly luciferase,and mice are imaged for bioluminescence. Alternatively, therapeuticefficacy and specificity of a single injection of CAR⁺ T cells in Nalm-6xenograft model can be measured as the following: NSG mice are injectedwith Nalm-6 transduced to stably express firefly luciferase, followed bya single tail-vein injection of T cells electroporated with CD33 CAR 7days later. Animals are imaged at various time points post injection.For example, photon-density heat maps of firefly luciferase positiveleukemia in representative mice at day 5 (2 days before treatment) andday 8 (24 hr post CAR⁺ PBLs) can be generated. Other assays, includingthose described in the Example section herein as well as those that areknown in the art can also be used to evaluate the CD33 CAR constructs ofthe invention.

Alternatively, or in combination to the methods disclosed herein,methods and compositions for one or more of: detection and/orquantification of CAR-expressing cells (e.g., in vitro or in vivo (e.g.,clinical monitoring)); immune cell expansion and/or activation; and/orCAR-specific selection, that involve the use of a CAR ligand, aredisclosed. In one exemplary embodiment, the CAR ligand is an antibodythat binds to the CAR molecule, e.g., binds to the extracellular antigenbinding domain of CAR (e.g., an antibody that binds to the antigenbinding domain, e.g., an anti-idiotypic antibody; or an antibody thatbinds to a constant region of the extracellular binding domain). Inother embodiments, the CAR ligand is a CAR antigen molecule (e.g., a CARantigen molecule as described herein).

In one aspect, a method for detecting and/or quantifying CAR-expressingcells is disclosed. For example, the CAR ligand can be used to detectand/or quantify CAR-expressing cells in vitro or in vivo (e.g., clinicalmonitoring of CAR-expressing cells in a patient, or dosing a patient).The method includes:

providing the CAR ligand (optionally, a labelled CAR ligand, e.g., a CARligand that includes a tag, a bead, a radioactive or fluorescent label);

acquiring the CAR-expressing cell (e.g., acquiring a sample containingCAR-expressing cells, such as a manufacturing sample or a clinicalsample);

contacting the CAR-expressing cell with the CAR ligand under conditionswhere binding occurs, thereby detecting the level (e.g., amount) of theCAR-expressing cells present. Binding of the CAR-expressing cell withthe CAR ligand can be detected using standard techniques such as FACS,ELISA and the like.

In another aspect, a method of expanding and/or activating cells (e.g.,immune effector cells) is disclosed. The method includes:

providing a CAR-expressing cell (e.g., a first CAR-expressing cell or atransiently expressing CAR cell);

contacting said CAR-expressing cell with a CAR ligand, e.g., a CARligand as described herein), under conditions where immune cellexpansion and/or proliferation occurs, thereby producing the activatedand/or expanded cell population.

In certain embodiments, the CAR ligand is present on (e.g., isimmobilized or attached to a substrate, e.g., a non-naturally occurringsubstrate). In some embodiments, the substrate is a non-cellularsubstrate. The non-cellular substrate can be a solid support chosenfrom, e.g., a plate (e.g., a microtiter plate), a membrane (e.g., anitrocellulose membrane), a matrix, a chip or a bead. In embodiments,the CAR ligand is present in the substrate (e.g., on the substratesurface). The CAR ligand can be immobilized, attached, or associatedcovalently or non-covalently (e.g., cross-linked) to the substrate. Inone embodiment, the CAR ligand is attached (e.g., covalently attached)to a bead. In the aforesaid embodiments, the immune cell population canbe expanded in vitro or ex vivo. The method can further includeculturing the population of immune cells in the presence of the ligandof the CAR molecule, e.g., using any of the methods described herein.

In other embodiments, the method of expanding and/or activating thecells further comprises addition of a second stimulatory molecule, e.g.,CD28. For example, the CAR ligand and the second stimulatory moleculecan be immobilized to a substrate, e.g., one or more beads, therebyproviding increased cell expansion and/or activation.

In yet another aspect, a method for selecting or enriching for a CARexpressing cell is provided. The method includes contacting the CARexpressing cell with a CAR ligand as described herein; and selecting thecell on the basis of binding of the CAR ligand.

In yet other embodiments, a method for depleting, reducing and/orkilling a CAR expressing cell is provided. The method includescontacting the CAR expressing cell with a CAR ligand as describedherein; and targeting the cell on the basis of binding of the CARligand, thereby reducing the number, and/or killing, the CAR-expressingcell. In one embodiment, the CAR ligand is coupled to a toxic agent(e.g., a toxin or a cell ablative drug). In another embodiment, theanti-idiotypic antibody can cause effector cell activity, e.g., ADCC orADC activities.

Exemplary anti-CAR antibodies that can be used in the methods disclosedherein are described, e.g., in WO 2014/190273 and by Jena et al.,“Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to DetectCD19-Specific T cells in Clinical Trials”, PLOS March 2013 8:3 e57838,the contents of which are incorporated by reference. In one embodiment,the anti-idiotypic antibody molecule recognizes an anti-CD19 antibodymolecule, e.g., an anti-CD19 scFv. For instance, the anti-idiotypicantibody molecule can compete for binding with the CD19-specific CAR mAbclone no. 136.20.1 described in Jena et al., PLOS March 2013 8:3 e57838;may have the same CDRs (e.g., one or more of, e.g., all of, VH CDR1, VHCDR2, CH CDR3, VL CDR1, VL CDR2, and VL CDR3, using the Kabatdefinition, the Chothia definition, or a combination of the Kabat andChothia definitions) as the CD19-specific CAR mAb clone no. 136.20.1;may have one or more (e.g., 2) variable regions as the CD19-specific CARmAb clone no. 136.20.1, or may comprise the CD19-specific CAR mAb cloneno. 136.20.1. In some embodiments, the anti-idiotypic antibody was madeaccording to a method described in Jena et al. In another embodiment,the anti-idiotypic antibody molecule is an anti-idiotypic antibodymolecule described in WO 2014/190273. In some embodiments, theanti-idiotypic antibody molecule has the same CDRs (e.g., one or moreof, e.g., all of, VH CDR1, VH CDR2, CH CDR3, VL CDR1, VL CDR2, and VLCDR3) as an antibody molecule of WO 2014/190273 such as 136.20.1; mayhave one or more (e.g., 2) variable regions of an antibody molecule ofWO 2014/190273, or may comprise an antibody molecule of WO 2014/190273such as 136.20.1. In other embodiments, the anti-CAR antibody binds to aconstant region of the extracellular binding domain of the CAR molecule,e.g., as described in WO 2014/190273. In some embodiments, the anti-CARantibody binds to a constant region of the extracellular binding domainof the CAR molecule, e.g., a heavy chain constant region (e.g., aCH2-CH3 hinge region) or light chain constant region. For instance, insome embodiments the anti-CAR antibody competes for binding with the 2D3monoclonal antibody described in WO 2014/190273, has the same CDRs(e.g., one or more of, e.g., all of, VH CDR1, VH CDR2, CH CDR3, VL CDR1,VL CDR2, and VL CDR3) as 2D3, or has one or more (e.g., 2) variableregions of 2D3, or comprises 2D3 as described in WO 2014/190273.

In some aspects and embodiments, the compositions and methods herein areoptimized for a specific subset of T cells, e.g., as described in U.S.Ser. No. 62/031,699 filed Jul. 31, 2014, the contents of which areincorporated herein by reference in their entirety. In some embodiments,the optimized subsets of T cells display an enhanced persistencecompared to a control T cell, e.g., a T cell of a different type (e.g.,CD8⁺ or CD4⁺) expressing the same construct.

In some embodiments, a CD4⁺ T cell comprises a CAR described herein,which CAR comprises an intracellular signaling domain suitable for(e.g., optimized for, e.g., leading to enhanced persistence in) a CD4⁺ Tcell, e.g., an ICOS domain. In some embodiments, a CD8⁺ T cell comprisesa CAR described herein, which CAR comprises an intracellular signalingdomain suitable for (e.g., optimized for, e.g., leading to enhancedpersistence of) a CD8⁺ T cell, e.g., a 4-1BB domain, a CD28 domain, oranother costimulatory domain other than an ICOS domain. In someembodiments, the CAR described herein comprises an antigen bindingdomain described herein, e.g., a CAR comprising an antigen bindingdomain that targets CD33, e.g., a CAR of Table 2 or a CAR having anamino acid sequence of SEQ ID NO: 140, or an antigen binding domaincomprising an amino acid sequence of SEQ ID NO: 147).

In an aspect, described herein is a method of treating a subject, e.g.,a subject having cancer. The method includes administering to saidsubject, an effective amount of:

1) a CD4⁺ T cell comprising a CAR (the CAR^(CD4+))

comprising:

an antigen binding domain, e.g., an antigen binding domain describedherein, e.g., an antigen binding domain that targets CD33], e.g., anantigen-binding domain of Table 2 or 9, or an antigen binding domaincomprising an amino acid sequence of SEQ ID NO: 140 or 147;

a transmembrane domain; and

an intracellular signaling domain, e.g., a first costimulatory domain,e.g., an ICOS domain; and

2) a CD8⁺ T cell comprising a CAR (the CAR^(cD8+)) comprising:

an antigen binding domain, e.g., an antigen binding domain describedherein, e.g., an antigen binding domain that targets CD33, e.g., anantigen-binding domain of Table 2 or 9, or an antigen binding domaincomprising an amino acid sequence of SEQ ID NO: 140 or 147;

a transmembrane domain; and

an intracellular signaling domain, e.g., a second costimulatory domain,e.g., a 4-1BB domain, a CD28 domain, or another costimulatory domainother than an ICOS domain;

wherein the CAR^(CD4+) and the CAR^(CD8+) differ from one another.

Optionally, the method further includes administering:

3) a second CD8+ T cell comprising a CAR (the second CAR^(CD8+))comprising:

an antigen binding domain, e.g., an antigen binding domain describedherein, e.g., an antigen binding domain that binds specifically to CD33,e.g., an antigen-binding domain of Table 2 or 9, or an antigen bindingdomain comprising an amino acid sequence of SEQ ID NO: 140 or 147;

a transmembrane domain; and

an intracellular signaling domain, wherein the second CAR^(CD8+)comprises an intracellular signaling domain, e.g., a costimulatorysignaling domain, not present on the CAR^(CD8+), and, optionally, doesnot comprise an ICOS signaling domain.

Therapeutic Application

CD33 Associated Diseases and/or Disorders

The present invention provides, among other things, compositions andmethods for treating a disease associated with expression of CD33 orcondition associated with cells which express CD33 including, e.g., aproliferative disease such as a cancer or malignancy or a precancerouscondition such as a myelodysplasia, a myelodysplastic syndrome or apreleukemia; or a noncancer related indication associated with cellswhich express CD33. In one aspect, a cancer associated with expressionof CD33 is a hematological cancer. In one aspect, a hematological cancerincludes but is not limited to AML, myelodysplastic syndrome, ALL,chronic myeloid leukemia, blastic plasmacytoid dendritic cell neoplasm,myeloproliferative neoplasms and the like. Further disease associatedwith expression of CD33 expression include, but are not limited to,e.g., atypical and/or non-classical cancers, malignancies, precancerousconditions or proliferative diseases associated with expression of CD33.Non-cancer related indications associated with expression of CD33 mayalso be included.

In one aspect, the invention provides methods for treating a diseaseassociated with CD33 expression. In one aspect, the invention providesmethods for treating a disease wherein part of the tumor is negative forCD33 and part of the tumor is positive for CD33. For example, the CAR ofthe invention is useful for treating subjects that have undergonetreatment for a disease associated with elevated expression of CD33,wherein the subject that has undergone treatment for elevated levels ofCD33 exhibits a disease associated with elevated levels of CD33. Inembodiments, the CAR of the invention is useful for treating subjectsthat have undergone treatment for a disease associated with expressionof CD33, wherein the subject that has undergone treatment related toexpression of CD33 exhibits a disease associated with expression ofCD33.

In one aspect, the invention pertains to a vector comprising CD33 CARoperably linked to promoter for expression in mammalian immune effectorcells, e.g., T cells or NK cells. In one aspect, the invention providesa recombinant immune effector cell (e.g., T cell or NK cell) expressingthe CD33 CAR for use in treating CD33-expressing tumors, wherein therecombinant immune effector cell (e.g., T cell or NK cell) expressingthe CD33 CAR is termed a CD33 CAR-expressing cell (e.g., CD33 CART orCD33 CAR-expressing NK cell). In one aspect, the CAR-expressing cell(e.g., CD33 CART or CD33 CAR-expressing NK cell) of the invention iscapable of contacting a tumor cell with at least one CD33 CAR of theinvention expressed on its surface such that the CAR-expressing cell(e.g., CD33 CART or CD33 CAR-expressing NK cell) targets the tumor celland growth of the tumor is inhibited.

In one aspect, the invention pertains to a method of inhibiting growthof a CD33-expressing tumor cell, comprising contacting the tumor cellwith a CD33 CAR-expressing cell (e.g., CD33 CART or CD33 CAR-expressingNK cell) of the present invention such that the CAR-expressing cell(e.g., CD33 CART or CD33 CAR-expressing NK cell) is activated inresponse to the antigen and targets the cancer cell, wherein the growthof the tumor is inhibited.

In one aspect, the invention pertains to a method of treating cancer ina subject. The method comprises administering to the subject a CD33CAR-expressing cell (e.g., CD33 CART or CD33 CAR-expressing NK cell) ofthe present invention such that the cancer is treated in the subject. Anexample of a cancer that is treatable by the CD33 CAR-expressing cell(e.g., CD33 CART or CD33 CAR-expressing NK cell) of the invention is acancer associated with expression of CD33. An example of a cancer thatis treatable by the CD33 CAR-expressing cell (e.g., CD33 CART or CD33CAR-expressing NK cell) of the invention includes but is not limited toAML, myelodysplastic syndrome, Chronic myeloid leukemia and othermyeloproliferative neoplasms, or Blastic plasmacytoid dendritic cellneoplasm, and the like.

The invention includes a type of cellular therapy where immune effectorcells, e.g., T cells or NK cells, are genetically modified to express achimeric antigen receptor (CAR) and the CAR-expressing cell (e.g., CD33CART or CD33 CAR-expressing NK cell) is infused to a recipient in needthereof. The infused cell is able to kill tumor cells in the recipient.Unlike antibody therapies, CAR-modified cells (e.g., T cells or NKcells) are able to replicate in vivo resulting in long-term persistencethat can lead to sustained tumor control. In various aspects, the cells(e.g., T cells or NK cells) administered to the patient, or theirprogeny, persist in the patient for at least four months, five months,six months, seven months, eight months, nine months, ten months, elevenmonths, twelve months, thirteen months, fourteen month, fifteen months,sixteen months, seventeen months, eighteen months, nineteen months,twenty months, twenty-one months, twenty-two months, twenty-threemonths, two years, three years, four years, or five years afteradministration of the cell (e.g., T cell or NK cell) to the patient.

The invention also includes a type of cellular therapy where immuneeffector cells (e.g., T cells or NK cells) are modified, e.g., by invitro transcribed RNA, to transiently express a chimeric antigenreceptor (CAR) and the immune effector cell (e.g., T cell or NK cell) isinfused to a recipient in need thereof. The infused cell is able to killtumor cells in the recipient. Thus, in various aspects, the immuneeffector cells (e.g., T cells or NK cells) administered to the patient,is present for less than one month, e.g., three weeks, two weeks, oneweek, after administration of the immune effector cell (e.g., T cell orNK cell) to the patient.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the CAR-modified immune effector cells(e.g., T cells or NK cells) may be an active or a passive immuneresponse, or alternatively may be due to a direct vs indirect immuneresponse. In one aspect, the CAR transduced immune effector cells (e.g.,T cells or NK cells) exhibit specific proinflammatory cytokine secretionand potent cytolytic activity in response to human cancer cellsexpressing CD33, resist soluble CD33 inhibition, mediate bystanderkilling and mediate regression of an established human tumor. Forexample, antigen-less tumor cells within a heterogeneous field ofCD33-expressing tumor may be susceptible to indirect destruction byCD33-redirected immune effector cells (e.g., T cells or NK cells) thathas previously reacted against adjacent antigen-positive cancer cells.

In one aspect, the fully-human CAR-modified immune effector cells (e.g.,T cells or NK cells) of the invention may be a type of vaccine for exvivo immunization and/or in vivo therapy in a mammal. In one aspect, themammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (e.g., a human)and genetically modified (i.e., transduced or transfected in vitro) witha vector expressing a CAR disclosed herein. The CAR-modified cell can beadministered to a mammalian recipient to provide a therapeutic benefit.The mammalian recipient may be a human and the CAR-modified cell can beautologous with respect to the recipient. Alternatively, the cells canbe allogeneic, syngeneic or xenogeneic with respect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modifiedimmune effector cells (e.g., T cells or NK cells) of the invention areused in the treatment of diseases, disorders and conditions associatedwith expression of CD33. In certain aspects, the cells of the inventionare used in the treatment of patients at risk for developing diseases,disorders and conditions associated with expression of CD33. Thus, thepresent invention provides methods for the treatment or prevention ofdiseases, disorders and conditions associated with expression of CD33comprising administering to a subject in need thereof, a therapeuticallyeffective amount of the CAR-modified immune effector cells (e.g., Tcells or NK cells) of the invention.

In one aspect the CAR-expressing cells (e.g., CART cells orCAR-expressing NK cells) of the inventions may be used to treat aproliferative disease such as a cancer or malignancy or is aprecancerous condition such as a myelodysplasia, a myelodysplasticsyndrome or a preleukemia. In one aspect, a cancer associated withexpression of CD33 is a hematological cancerpreleukemiahyperproliferative disorder, hyperplasia or a dysplasia,which is characterized by abnormal growth of cells.

In one aspect, the CAR-expressing cells (e.g., CART cells orCAR-expressing NK cells) of the invention are used to treat a cancer,wherein the cancer is a hematological cancer. Hematological cancerconditions are the types of cancer such as leukemia and malignantlymphoproliferative conditions that affect blood, bone marrow and thelymphatic system.

In one aspect, the compositions and CAR-expressing cells (e.g., CARTcells or CAR-expressing NK cells) of the present invention areparticularly useful for treating myeloid leukemias, AML and itssubtypes, chronic myeloid leukemia (CML), and myelodysplastic syndrome(MDS).

Leukemia can be classified as acute leukemia and chronic leukemia. Acuteleukemia can be further classified as acute myelogenous leukemia (AML)and acute lymphoid leukemia (ALL). Chronic leukemia includes chronicmyelogenous leukemia (CML) and chronic lymphoid leukemia (CLL). Otherrelated conditions include myelodysplastic syndromes (MDS, formerlyknown as “preleukemia”) which are a diverse collection of hematologicalconditions united by ineffective production (or dysplasia) of myeloidblood cells and risk of transformation to AML.

Lymphoma is a group of blood cell tumors that develop from lymphocytes.Exemplary lymphomas include non-Hodgkin lymphoma and Hodgkin lymphoma.

In AML, malignant transformation and uncontrolled proliferation of anabnormally differentiated, long-lived myeloid progenitor cell results inhigh circulating numbers of immature blood forms and replacement ofnormal marrow by malignant cells. Symptoms include fatigue, pallor, easybruising and bleeding, fever, and infection; symptoms of leukemicinfiltration are present in only about 5% of patients (often as skinmanifestations). Examination of peripheral blood smear and bone marrowis diagnostic. Existing treatment includes induction chemotherapy toachieve remission and post-remission chemotherapy (with or without stemcell transplantation) to avoid relapse.

AML has a number of subtypes that are distinguished from each other bymorphology, immunophenotype, and cytochemistry. Five classes aredescribed, based on predominant cell type, including myeloid,myeloid-monocytic, monocytic, erythroid, and megakaryocytic.

Remission induction rates range from 50 to 85%. Long-term disease-freesurvival reportedly occurs in 20 to 40% of patients and increases to 40to 50% in younger patients treated with stem cell transplantation.

Prognostic factors help determine treatment protocol and intensity;patients with strongly negative prognostic features are usually givenmore intense forms of therapy, because the potential benefits arethought to justify the increased treatment toxicity. The most importantprognostic factor is the leukemia cell karyotype; favorable karyotypesinclude t(15;17), t(8;21), and inv16 (p13; q22). Negative factorsinclude increasing age, a preceding myelodysplastic phase, secondaryleukemia, high WBC count, and absence of Auer rods.

Initial therapy attempts to induce remission and differs most from ALLin that AML responds to fewer drugs. The basic induction regimenincludes cytarabine by continuous IV infusion or high doses for 5 to 7days; daunorubicin or idarubicin is given IV for 3 days during thistime. Some regimens include 6-thioguanine, etoposide, vincristine, andprednisone, but their contribution is unclear. Treatment usually resultsin significant myelosuppression, with infection or bleeding; there issignificant latency before marrow recovery. During this time, meticulouspreventive and supportive care is vital.

Chronic myelogenous (or myeloid) leukemia (CML) is also known as chronicgranulocytic leukemia, and is characterized as a cancer of the whiteblood cells. Common treatment regimens for CML include Bcr-Abl tyrosinekinase inhibitors, imatinib (Gleevec®), dasatinib and nilotinib. Bcr-Abltyrosine kinase inhibitors are specifically useful for CML patients withthe Philadelphia chromosome translocation.

Myelodysplastic syndromes (MDS) are hematological medical conditionscharacterized by disorderly and ineffective hematopoiesis, or bloodproduction. Thus, the number and quality of blood-forming cells declineirreversibly. Some patients with MDS can develop severeanemia, whileothers are asymptomatic. The classification scheme for MDS is known inthe art, with criteria designating the ratio or frequency of particularblood cell types, e.g., myeloblasts, monocytes, and red cell precursors.MDS includes refractory anemia, refractory anemia with ringsideroblasts, refractory anemia with excess blasts, refractory anemiawith excess blasts in transformation, chronic myelomonocytic leukemia(CML).

Treatments for MDS vary with the severity of the symptoms. Aggressiveforms of treatment for patients experiencing severe symptoms includebone marrow transplants and supportive care with blood product support(e.g., blood transfusions) and hematopoietic growth factors (e.g.,erythropoietin). Other agents are frequently used to treat MDS:5-azacytidine, decitabine, and lenalidomide. In some cases, ironchelators deferoxamine (Desferal®) and deferasirox (Exjade®) may also beadministered.

In another embodiment, the CAR-expressing cells (e.g., CART cells orCAR-expressing NK cells) of the present invention are used to treatcancers or leukemias with leukemia stem cells. For example, the leukemiastem cells are CD34⁺/CD38⁻ leukemia cells.

The present invention provides, among other things, compositions andmethods for treating cancer. In one aspect, the cancer is a hematologiccancer including but is not limited to leukemia (such as acutemyelogenous leukemia, chronic myelogenous leukemia, acute lymphoidleukemia, chronic lymphoid leukemia and myelodysplastic syndrome) andmalignant lymphoproliferative conditions, including lymphoma (such asmultiple myeloma, non-Hodgkin

lymphoma, Burkitt

lymphoma, and small cell- and large cell-follicular lymphoma).

In one aspect, the CAR-expressing cells (e.g., CART cells orCAR-expressing NK cells) of the invention may be used to treat othercancers and malignancies such as, but not limited to, e.g., acuteleukemias including but not limited to, e.g., B-cell acute lymphoidleukemia (“BALL”), T-cell acute lymphoid leukemia (“TALL”), acutelymphoid leukemia (ALL); one or more chronic leukemias including but notlimited to, e.g., chronic myelogenous leukemia (CIVIL), chroniclymphocytic leukemia (CLL); additional hematologic cancers orhematologic conditions including, but not limited to, e.g., B cellprolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm,Burkitt

lymphoma, diffuse large B cell lymphoma, Follicular lymphoma, Hairy cellleukemia, small cell- or a large cell-follicular lymphoma, malignantlymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma,Marginal zone lymphoma, multiple myeloma, myelodysplasia andmyelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablasticlymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrommacroglobulinemia, and “preleukemia” which are a diverse collection ofhematological conditions united by ineffective production (or dysplasia)of myeloid blood cells, and the like. The CAR-modified immune effectorcells (e.g., T cells or NK cells) of the present invention may beadministered either alone, or as a pharmaceutical composition incombination with diluents and/or with other components such as IL-2 orother cytokines or cell populations.

The present invention also provides methods for inhibiting theproliferation or reducing a CD33-expressing cell population, the methodscomprising contacting a population of cells comprising a CD33-expressingcell with a CD33 CAR-expressing cell (e.g., CD33CART cell or CD33CAR-expressing NK cell) of the invention that binds to theCD33-expressing cell. In a specific aspect, the present inventionprovides methods for inhibiting the proliferation or reducing thepopulation of cancer cells expressing CD33, the methods comprisingcontacting the CD33-expressing cancer cell population with a CD33CAR-expressing cell (e.g., CD33CART cell or CD33 CAR-expressing NK cell)of the invention that binds to the CD33-expressing cell. In one aspect,the present invention provides methods for inhibiting the proliferationor reducing the population of cancer cells expressing CD33, the methodscomprising contacting the CD33-expressing cancer cell population with aCD33 CAR-expressing cell (e.g., CD33CART cell or CD33 CAR-expressing NKcell) of the invention that binds to the CD33-expressing cell. Incertain aspects, the CD33 CAR-expressing cell (e.g., CD33CART cell orCD33 CAR-expressing NK cell) of the invention reduces the quantity,number, amount or percentage of cells and/or cancer cells by at least25%, at least 30%, at least 40%, at least 50%, at least 65%, at least75%, at least 85%, at least 95%, or at least 99% in a subject with oranimal model for myeloid leukemia or another cancer associated withCD33-expressing cells relative to a negative control. In one aspect, thesubject is a human.

The present invention also provides methods for preventing, treatingand/or managing a disease associated with CD33-expressing cells (e.g., ahematologic cancer or atypical cancer expressing CD33), the methodscomprising administering to a subject in need a CD33 CAR-expressing cell(e.g., CD33CART cell or CD33 CAR-expressing NK cell) of the inventionthat binds to the CD33-expressing cell. In one aspect, the subject is ahuman. Non-limiting examples of disorders associated withCD33-expressing cells include autoimmune disorders (such as lupus),inflammatory disorders (such as allergies and asthma) and cancers (suchas hematological cancers or atypical cancers expressing CD33).

The present invention also provides methods for preventing, treatingand/or managing a disease associated with CD33-expressing cells, themethods comprising administering to a subject in need a CD33CAR-expressing cell (e.g., CD33CART cell or CD33 CAR-expressing NK cell)of the invention that binds to the CD33-expressing cell. In one aspect,the subject is a human.

The present invention provides methods for preventing relapse of cancerassociated with CD33-expressing cells, the methods comprisingadministering to a subject in need thereof a CD33 CAR-expressing cell(e.g., CD33CART cell or CD33 CAR-expressing NK cell) of the inventionthat binds to the CD33-expressing cell. In one aspect, the methodscomprise administering to the subject in need thereof an effectiveamount of a CD33 CAR-expressing cell (e.g., CD33CART cell or CD33CAR-expressing NK cell) described herein that binds to theCD33-expressing cell in combination with an effective amount of anothertherapy.

Combination Therapies

A CAR-expressing cell described herein may be used in combination withother known agents and therapies. Administered “in combination”, as usedherein, means that two (or more) different treatments are delivered tothe subject during the course of the subject affliction with thedisorder, e.g., the two or more treatments are delivered after thesubject has been diagnosed with the disorder and before the disorder hasbeen cured or eliminated or treatment has ceased for other reasons. Insome embodiments, the delivery of one treatment is still occurring whenthe delivery of the second begins, so that there is overlap in terms ofadministration. This is sometimes referred to herein as “simultaneous”or “concurrent delivery”. In other embodiments, the delivery of onetreatment ends before the delivery of the other treatment begins. Insome embodiments of either case, the treatment is more effective becauseof combined administration. For example, the second treatment is moreeffective, e.g., an equivalent effect is seen with less of the secondtreatment, or the second treatment reduces symptoms to a greater extent,than would be seen if the second treatment were administered in theabsence of the first treatment, or the analogous situation is seen withthe first treatment. In some embodiments, delivery is such that thereduction in a symptom, or other parameter related to the disorder isgreater than what would be observed with one treatment delivered in theabsence of the other. The effect of the two treatments can be partiallyadditive, wholly additive, or greater than additive. The delivery can besuch that an effect of the first treatment delivered is still detectablewhen the second is delivered.

A CAR-expressing cell described herein and the at least one additionaltherapeutic agent can be administered simultaneously, in the same or inseparate compositions, or sequentially. For sequential administration,the CAR-expressing cell described herein can be administered first, andthe additional agent can be administered second, or the order ofadministration can be reversed.

The CAR therapy and/or other therapeutic agents, procedures ormodalities can be administered during periods of active disorder, orduring a period of remission or less active disease. The CAR therapy canbe administered before the other treatment, concurrently with thetreatment, post-treatment, or during remission of the disorder.

When administered in combination, the CAR therapy and the additionalagent (e.g., second or third agent), or all, can be administered in anamount or dose that is higher, lower or the same than the amount ordosage of each agent used individually, e.g., as a monotherapy. Incertain embodiments, the administered amount or dosage of the CARtherapy, the additional agent (e.g., second or third agent), or all, islower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%)than the amount or dosage of each agent used individually, e.g., as amonotherapy. In other embodiments, the amount or dosage of the CARtherapy, the additional agent (e.g., second or third agent), or all,that results in a desired effect (e.g., treatment of cancer) is lower(e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower)than the amount or dosage of each agent used individually, e.g., as amonotherapy, required to achieve the same therapeutic effect.

In further aspects, a CAR-expressing cell described herein may be usedin a treatment regimen in combination with surgery, chemotherapy,radiation, immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies or otherantibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin,mycophenolic acid, steroids, FR901228, cytokines, and irradiation.peptide vaccine, such as that described in Izumoto et al. 2008 JNeurosurg 108:963-971.

In certain instances, compounds of the present invention are combinedwith other therapeutic agents, such as other anti-cancer agents,anti-allergic agents, anti-nausea agents (or anti-emetics), painrelievers, cytoprotective agents, and combinations thereof.

In one embodiment, a CAR-expressing cell described herein can be used incombination with a chemotherapeutic agent. Exemplary chemotherapeuticagents include an anthracycline (e.g., doxorubicin (e.g., liposomaldoxorubicin)). a vinca alkaloid (e.g., vinblastine, vincristine,vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide,decarbazine, melphalan, ifosfamide, temozolomide), an immune cellantibody (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab,tositumomab, brentuximab), an antimetabolite (including, e.g., folicacid antagonists, pyrimidine analogs, purine analogs and adenosinedeaminase inhibitors (e.g., fludarabine)), an mTOR inhibitor, a TNFRglucocorticoid induced TNFR related protein (GITR) agonist, a proteasomeinhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), animmunomodulator such as thalidomide or a thalidomide derivative (e.g.,lenalidomide).

General Chemotherapeutic agents considered for use in combinationtherapies include busulfan (Myleran®), busulfan injection (Busulfex®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®),fludarabine phosphate (Fludara®), hydroxyurea (Hydrea®), Idarubicin(Idamycin®), mitoxantrone (Novantrone®), Gemtuzumab Ozogamicin(Mylotarg®).

In embodiments, general chemotherapeutic agents considered for use incombination therapies include anastrozole (Arimidex®), bicalutamide(Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®),busulfan injection (Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®),etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil(Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®),ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®),leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine(Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®),mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate(Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine(Tirazone®), topotecan hydrochloride for injection (Hycamptin®),vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine(Navelbine®).

Anti-cancer agents of particular interest for combinations with thecompounds of the present invention include: anthracyclines; alkylatingagents; antimetabolites; drugs that inhibit either the calcium dependentphosphatase calcineurin or the p70S6 kinase FK506) or inhibit the p70S6kinase; mTOR inhibitors; immunomodulators; anthracyclines; vincaalkaloids; proteosome inhibitors; GITR agonists; protein tyrosinephosphatase inhibitors; a CDK4 kinase inhibitor; a BTK inhibitor; a MKNkinase inhibitor; a DGK kinase inhibitor; or an oncolytic virus.

Exemplary antimetabolites include, without limitation, pyrimidineanalogs, purine analogs and adenosine deaminase inhibitors):methotrexate (Rheumatrex®, Trexall®), 5-fluorouracil (Adrucil®, Efudex®,Fluoroplex®), floxuridine (FUDF®), cytarabine (Cytosar-U®, TarabinePFS), 6-mercaptopurine (Puri-Nethol®)), 6-thioguanine (ThioguanineTabloid®), fludarabine phosphate (Fludara®), pentostatin (Nipent®),pemetrexed (Alimta®), raltitrexed (Tomudex®), cladribine (Leustatin®),clofarabine (Clofarex®, Clolar®), azacitidine (Vidaza®), decitabine andgemcitabine (Gemzar®). Preferred antimetabolites include, cytarabine,clofarabine and fludarabine.

Exemplary alkylating agents include, without limitation, nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas andtriazenes): uracil mustard (Aminouracil Mustard®, Chlorethaminacil®,Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracilnitrogen Mustard®, Uracillost®, Uracilmostaza®, Uramustin®,Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®,Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide(Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman(Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®),triethylenethiophosphoramine, Temozolomide (Temodar®), thiotepa(Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®),lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine(DTIC-Dome®). Additional exemplary alkylating agents include, withoutlimitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® andTemodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®);Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard,Alkeran®); Altretamine (also known as hexamethylmelamine (HMM),Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan(Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (alsoknown as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® andPlatinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® andNeosar®); Dacarbazine (also known as DTIC, DIC and imidazolecarboxamide, DTIC-Dome®); Altretamine (also known as hexamethylmelamine(HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine(Matulane®); Mechlorethamine (also known as nitrogen mustard, mustineand mechloroethamine hydrochloride, Mustargen®); Streptozocin(Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA,Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®,Revimmune®); and Bendamustine HCl (Treanda®).

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with fludarabine, cyclophosphamide, and/orrituximab. In embodiments, a CAR-expressing cell described herein isadministered to a subject in combination with fludarabine,cyclophosphamide, and rituximab (FCR). In embodiments, the subject hasCLL. For example, the subject has a deletion in the short arm ofchromosome 17 (del(17p), e.g., in a leukemic cell). In other examples,the subject does not have a del(17p). In embodiments, the subjectcomprises a leukemic cell comprising a mutation in the immunoglobulinheavy-chain variable-region (IgV_(H)) gene. In other embodiments, thesubject does not comprise a leukemic cell comprising a mutation in theimmunoglobulin heavy-chain variable-region (IgV_(H)) gene. Inembodiments, the fludarabine is administered at a dosage of about 10-50mg/m² (e.g., about 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or45-50 mg/m²), e.g., intravenously. In embodiments, the cyclophosphamideis administered at a dosage of about 200-300 mg/m² (e.g., about 200-225,225-250, 250-275, or 275-300 mg/m²), e.g., intravenously. Inembodiments, the rituximab is administered at a dosage of about 400-600mg/m2 (e.g., 400-450, 450-500, 500-550, or 550-600 mg/m²), e.g.,intravenously.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with bendamustine and rituximab. Inembodiments, the subject has CLL. For example, the subject has adeletion in the short arm of chromosome 17 (del(17p), e.g., in aleukemic cell). In other examples, the subject does not have a del(17p).In embodiments, the subject comprises a leukemic cell comprising amutation in the immunoglobulin heavy-chain variable-region (IgV_(H))gene. In other embodiments, the subject does not comprise a leukemiccell comprising a mutation in the immunoglobulin heavy-chainvariable-region (IgV_(H)) gene. In embodiments, the bendamustine isadministered at a dosage of about 70-110 mg/m2 (e.g., 70-80, 80-90,90-100, or 100-110 mg/m2), e.g., intravenously. In embodiments, therituximab is administered at a dosage of about 400-600 mg/m2 (e.g.,400-450, 450-500, 500-550, or 550-600 mg/m²), e.g., intravenously.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with rituximab, cyclophosphamide,doxorubicine, vincristine, and/or a corticosteroid (e.g., prednisone).In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with rituximab, cyclophosphamide,doxorubicine, vincristine, and prednisone (R-CHOP). In embodiments, thesubject has diffuse large B-cell lymphoma (DLBCL). In embodiments, thesubject has nonbulky limited-stage DLBCL (e.g., comprises a tumor havinga size/diameter of less than 7 cm). In embodiments, the subject istreated with radiation in combination with the R-CHOP. For example, thesubject is administered R-CHOP (e.g., 1-6 cycles, e.g., 1, 2, 3, 4, 5,or 6 cycles of R-CHOP), followed by radiation. In some cases, thesubject is administered R-CHOP (e.g., 1-6 cycles, e.g., 1, 2, 3, 4, 5,or 6 cycles of R-CHOP) following radiation.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with etoposide, prednisone, vincristine,cyclophosphamide, doxorubicin, and/or rituximab. In embodiments, aCAR-expressing cell described herein is administered to a subject incombination with etoposide, prednisone, vincristine, cyclophosphamide,doxorubicin, and rituximab (EPOCH-R). In embodiments, a CAR-expressingcell described herein is administered to a subject in combination withdose-adjusted EPOCH-R (DA-EPOCH-R). In embodiments, the subject has a Bcell lymphoma, e.g., a Myc-rearranged aggressive B cell lymphoma.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with rituximab and/or lenalidomide.Lenalidomide ((RS)-3-(4-Amino-1-oxo1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione) is animmunomodulator. In embodiments, a CAR-expressing cell described hereinis administered to a subject in combination with rituximab andlenalidomide. In embodiments, the subject has follicular lymphoma (FL)or mantle cell lymphoma (MCL). In embodiments, the subject has FL andhas not previously been treated with a cancer therapy. In embodiments,lenalidomide is administered at a dosage of about 10-20 mg (e.g., 10-15or 15-20 mg), e.g., daily. In embodiments, rituximab is administered ata dosage of about 350-550 mg/m² (e.g., 350-375, 375-400, 400-425,425-450, 450-475, or 475-500 mg/m²), e.g., intravenously.

Exemplary mTOR inhibitors include, e.g., temsirolimus; ridaforolimus(formally known as deferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described inPCT Publication No. WO 03/064383); everolimus (Afinitor® or RAD001);rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3);emsirolimus,(5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502, CAS 1013101-36-4); andN²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-(SEQ ID NO: 378), inner salt (SF1126, CAS 936487-67-1), and XL765.

Exemplary immunomodulators include, e.g., afutuzumab (available fromRoche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®);thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (mixture of humancytokines including interleukin 1, interleukin 2, and interferon γ, CAS951209-71-5, available from IRX Therapeutics).

Exemplary anthracyclines include, e.g., doxorubicin (Adriamycin® andRubex®); bleomycin (Lenoxane®); daunorubicin (dauorubicin hydrochloride,daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicinliposomal (daunorubicin citrate liposome, DaunoXome®); mitoxantrone(DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®,Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin;ravidomycin; and desacetylravidomycin.

Exemplary vinca alkaloids include, e.g., vinorelbine tartrate(Navelbine®), Vincristine (Oncovin®), and Vindesine (Eldisine®));vinblastine (also known as vinblastine sulfate, vincaleukoblastine andVLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®).

Exemplary proteosome inhibitors include bortezomib (Velcade®);carfilzomib (PX-171-007,(S)-4-Methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-pentanamide);marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib(CEP-18770); andO-Methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide(ONX-0912).

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with brentuximab. Brentuximab is anantibody-drug conjugate of anti-CD30 antibody and monomethyl auristatinE. In embodiments, the subject has Hodgkin's lymphoma (HL), e.g.,relapsed or refractory HL. In embodiments, the subject comprises CD30+HL. In embodiments, the subject has undergone an autologous stem celltransplant (ASCT). In embodiments, the subject has not undergone anASCT. In embodiments, brentuximab is administered at a dosage of about1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg), e.g.,intravenously, e.g., every 3 weeks.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with brentuximab and dacarbazine or incombination with brentuximab and bendamustine. Dacarbazine is analkylating agent with a chemical name of5-(3,3-Dimethyl-1-triazenyl)imidazole-4-carboxamide. Bendamustine is analkylating agent with a chemical name of4-[5-[Bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid.In embodiments, the subject has Hodgkin's lymphoma (HL). In embodiments,the subject has not previously been treated with a cancer therapy. Inembodiments, the subject is at least 60 years of age, e.g., 60, 65, 70,75, 80, 85, or older. In embodiments, dacarbazine is administered at adosage of about 300-450 mg/m² (e.g., about 300-325, 325-350, 350-375,375-400, 400-425, or 425-450 mg/m²), e.g., intravenously. Inembodiments, bendamustine is administered at a dosage of about 75-125mg/m2 (e.g., 75-100 or 100-125 mg/m², e.g., about 90 mg/m²), e.g.,intravenously. In embodiments, brentuximab is administered at a dosageof about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg),e.g., intravenously, e.g., every 3 weeks.

In some embodiments, a CAR-expressing cell described herein isadministered to a subject in combination with a CD20 inhibitor, e.g., ananti-CD20 antibody (e.g., an anti-CD20 mono- or bispecific antibody) ora fragment thereof. Exemplary anti-CD20 antibodies include but are notlimited to rituximab, ofatumumab, ocrelizumab, veltuzumab, obinutuzumab,TRU-015 (Trubion Pharmaceuticals), ocaratuzumab, and Pro131921(Genentech). See, e.g., Lim et al. Haematologica. 95.1(2010):135-43.

In some embodiments, the anti-CD20 antibody comprises rituximab.Rituximab is a chimeric mouse/human monoclonal antibody IgG1 kappa thatbinds to CD20 and causes cytolysis of a CD20 expressing cell, e.g., asdescribed inwww.accessdata.fda.gov/drugsatfda_docs/label/2010/103705s5311lbl.pdf. Inembodiments, a CAR-expressing cell described herein is administered to asubject in combination with rituximab. In embodiments, the subject hasCLL or SLL.

In some embodiments, rituximab is administered intravenously, e.g., asan intravenous infusion. For example, each infusion provides about500-2000 mg (e.g., about 500-550, 550-600, 600-650, 650-700, 700-750,750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200,1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800,1800-1900, or 1900-2000 mg) of rituximab. In some embodiments, rituximabis administered at a dose of 150 mg/m² to 750 mg/m², e.g., about 150-175mg/m², 175-200 mg/m², 200-225 mg/m², 225-250 mg/m², 250-300 mg/m²,300-325 mg/m², 325-350 mg/m², 350-375 mg/m², 375-400 mg/m², 400-425mg/m², 425-450 mg/m², 450-475 mg/m², 475-500 mg/m², 500-525 mg/m²,525-550 mg/m², 550-575 mg/m², 575-600 mg/m², 600-625 mg/m², 625-650mg/m², 650-675 mg/m², or 675-700 mg/m², where m² indicates the bodysurface area of the subject. In some embodiments, rituximab isadministered at a dosing interval of at least 4 days, e.g., 4, 7, 14,21, 28, 35 days, or more. For example, rituximab is administered at adosing interval of at least 0.5 weeks, e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8weeks, or more. In some embodiments, rituximab is administered at a doseand dosing interval described herein for a period of time, e.g., atleast 2 weeks, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 weeks, or greater. For example, rituximab isadministered at a dose and dosing interval described herein for a totalof at least 4 doses per treatment cycle (e.g., at least 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, or more doses per treatment cycle).

In some embodiments, the anti-CD20 antibody comprises ofatumumab.Ofatumumab is an anti-CD20 IgG1κ human monoclonal antibody with amolecular weight of approximately 149 kDa. For example, ofatumumab isgenerated using transgenic mouse and hybridoma technology and isexpressed and purified from a recombinant murine cell line (NS0). See,e.g., www.accessdata.fda.gov/drugsatfda_docs/label/2009/125326lbl.pdf;and Clinical Trial Identifier number NCT01363128, NCT01515176,NCT01626352, and NCT01397591. In embodiments, a CAR-expressing celldescribed herein is administered to a subject in combination withofatumumab. In embodiments, the subject has CLL or SLL.

In some embodiments, ofatumumab is administered as an intravenousinfusion. For example, each infusion provides about 150-3000 mg (e.g.,about 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500,500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900,900-950, 950-1000, 1000-1200, 1200-1400, 1400-1600, 1600-1800,1800-2000, 2000-2200, 2200-2400, 2400-2600, 2600-2800, or 2800-3000 mg)of ofatumumab. In embodiments, ofatumumab is administered at a startingdosage of about 300 mg, followed by 2000 mg, e.g., for about 11 doses,e.g., for 24 weeks. In some embodiments, ofatumumab is administered at adosing interval of at least 4 days, e.g., 4, 7, 14, 21, 28, 35 days, ormore. For example, ofatumumab is administered at a dosing interval of atleast 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 26, 28,20, 22, 24, 26, 28, 30 weeks, or more. In some embodiments, ofatumumabis administered at a dose and dosing interval described herein for aperiod of time, e.g., at least 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 40, 50,60 weeks or greater, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months orgreater, or 1, 2, 3, 4, 5 years or greater. For example, ofatumumab isadministered at a dose and dosing interval described herein for a totalof at least 2 doses per treatment cycle (e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, or more doses per treatmentcycle).

In some cases, the anti-CD20 antibody comprises ocrelizumab. Ocrelizumabis a humanized anti-CD20 monoclonal antibody, e.g., as described inClinical Trials Identifier Nos. NCT00077870, NCT01412333, NCT00779220,NCT00673920, NCT01194570, and Kappos et al. Lancet.19.378(2011):1779-87.

In some cases, the anti-CD20 antibody comprises veltuzumab. Veltuzumabis a humanized monoclonal antibody against CD20. See, e.g., ClinicalTrial Identifier No. NCT00547066, NCT00546793, NCT01101581, andGoldenberg et al. Leuk Lymphoma. 51(5)(2010):747-55.

In some cases, the anti-CD20 antibody comprises GA101. GA101 (alsocalled obinutuzumab or R05072759) is a humanized and glyco-engineeredanti-CD20 monoclonal antibody. See, e.g., Robak. Curr. Opin. Investig.Drugs. 10.6(2009):588-96; Clinical Trial Identifier Numbers:NCT01995669, NCT01889797, NCT02229422, and NCT01414205; andwww.accessdata.fda.gov/drugsatfda_docs/label/2013/125486s000lbl.pdf.

In some cases, the anti-CD20 antibody comprises AME-133v. AME-133v (alsocalled LY2469298 or ocaratuzumab) is a humanized IgG1 monoclonalantibody against CD20 with increased affinity for the FcγRIIIa receptorand an enhanced antibody dependent cellular cytotoxicity (ADCC) activitycompared with rituximab. See, e.g., Robak et al. BioDrugs25.1(2011):13-25; and Forero-Torres et al. Clin Cancer Res.18.5(2012):1395-403.

In some cases, the anti-CD20 antibody comprises PRO131921. PRO131921 isa humanized anti-CD20 monoclonal antibody engineered to have betterbinding to FcγRIIIa and enhanced ADCC compared with rituximab. See,e.g., Robak et al. BioDrugs 25.1(2011):13-25; and Casulo et al. ClinImmunol. 154.1(2014):37-46; and Clinical Trial Identifier No.NCT00452127.

In some cases, the anti-CD20 antibody comprises TRU-015. TRU-015 is ananti-CD20 fusion protein derived from domains of an antibody againstCD20. TRU-015 is smaller than monoclonal antibodies, but retainsFc-mediated effector functions. See, e.g., Robak et al. BioDrugs25.1(2011):13-25. TRU-015 contains an anti-CD20 single-chain variablefragment (scFv) linked to human IgG1 hinge, CH2, and CH3 domains butlacks CH1 and CL domains.

In some embodiments, an anti-CD20 antibody described herein isconjugated or otherwise bound to a therapeutic agent, e.g., achemotherapeutic agent (e.g., cytoxan, fludarabine, histone deacetylaseinhibitor, demethylating agent, peptide vaccine, anti-tumor antibiotic,tyrosine kinase inhibitor, alkylating agent, anti-microtubule oranti-mitotic agent), anti-allergic agent, anti-nausea agent (oranti-emetic), pain reliever, or cytoprotective agent described herein.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a B-cell lymphoma 2 (BCL-2) inhibitor(e.g., venetoclax, also called ABT-199 or GDC-0199;) and/or rituximab.In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with venetoclax and rituximab. Venetoclax isa small molecule that inhibits the anti-apoptotic protein, BCL-2. Thestructure of venetoclax(4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide)is shown below.

In embodiments, the subject has CLL. In embodiments, the subject hasrelapsed CLL, e.g., the subject has previously been administered acancer therapy. In embodiments, venetoclax is administered at a dosageof about 15-600 mg (e.g., 15-20, 20-50, 50-75, 75-100, 100-200, 200-300,300-400, 400-500, or 500-600 mg), e.g., daily. In embodiments, rituximabis administered at a dosage of about 350-550 mg/m2 (e.g., 350-375,375-400, 400-425, 425-450, 450-475, or 475-500 mg/m2), e.g.,intravenously, e.g., monthly.

In some embodiments, a CAR-expressing cell described herein isadministered in combination with an oncolytic virus. In embodiments,oncolytic viruses are capable of selectively replicating in andtriggering the death of or slowing the growth of a cancer cell. In somecases, oncolytic viruses have no effect or a minimal effect onnon-cancer cells. An oncolytic virus includes but is not limited to anoncolytic adenovirus, oncolytic Herpes Simplex Viruses, oncolyticretrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolyticSinbis virus, oncolytic influenza virus, or oncolytic RNA virus (e.g.,oncolytic reovirus, oncolytic Newcastle Disease Virus (NDV), oncolyticmeasles virus, or oncolytic vesicular stomatitis virus (VSV)).

In some embodiments, the oncolytic virus is a virus, e.g., recombinantoncolytic virus, described in US2010/0178684 A1, which is incorporatedherein by reference in its entirety. In some embodiments, a recombinantoncolytic virus comprises a nucleic acid sequence (e.g., heterologousnucleic acid sequence) encoding an inhibitor of an immune orinflammatory response, e.g., as described in US2010/0178684 A1,incorporated herein by reference in its entirety. In embodiments, therecombinant oncolytic virus, e.g., oncolytic NDV, comprises apro-apoptotic protein (e.g., apoptin), a cytokine (e.g., GM-CSF,interferon-gamma, interleukin-2 (IL-2), tumor necrosis factor-alpha), animmunoglobulin (e.g., an antibody against ED-B firbonectin), tumorassociated antigen, a bispecific adapter protein (e.g., bispecificantibody or antibody fragment directed against NDV HN protein and a Tcell co-stimulatory receptor, such as CD3 or CD28; or fusion proteinbetween human IL-2 and single chain antibody directed against NDV HNprotein). See, e.g., Zamarin et al. Future Microbiol. 7.3(2012):347-67,incorporated herein by reference in its entirety. In some embodiments,the oncolytic virus is a chimeric oncolytic NDV described in U.S. Pat.No. 8,591,881 B2, US 2012/0122185 A1, or US 2014/0271677 A1, each ofwhich is incorporated herein by reference in their entireties.

In some embodiments, the oncolytic virus comprises a conditionallyreplicative adenovirus (CRAd), which is designed to replicateexclusively in cancer cells. See, e.g., Alemany et al. NatureBiotechnol. 18(2000):723-27. In some embodiments, an oncolyticadenovirus comprises one described in Table 1 on page 725 of Alemany etal., incorporated herein by reference in its entirety.

Exemplary oncolytic viruses include but are not limited to thefollowing:

Group B Oncolytic Adenovirus (ColoAd1) (PsiOxus Therapeutics Ltd.) (see,e.g., Clinical Trial Identifier: NCT02053220);

ONCOS-102 (previously called CGTG-102), which is an adenoviruscomprising granulocyte-macrophage colony stimulating factor (GM-CSF)(Oncos Therapeutics) (see, e.g., Clinical Trial Identifier:NCT01598129);

VCN-01, which is a genetically modified oncolytic human adenovirusencoding human PH20 hyaluronidase (VCN Biosciences, S.L.) (see, e.g.,Clinical Trial Identifiers: NCT02045602 and NCT02045589);

Conditionally Replicative Adenovirus ICOVIR-5, which is a virus derivedfrom wild-type human adenovirus serotype 5 (Had5) that has been modifiedto selectively replicate in cancer cells with a deregulatedretinoblastoma/E2F pathway (Institut Català d□Oncologia) (see, e.g.,Clinical Trial Identifier: NCT01864759);

Celyvir, which comprises bone marrow-derived autologous mesenchymal stemcells (MSCs) infected with ICOVIR5, an oncolytic adenovirus (HospitalInfantil Universitario Niño Jeśus, Madrid, Spain/Ramon Alemany) (see,e.g., Clinical Trial Identifier: NCT01844661);

CG0070, which is a conditionally replicating oncolytic serotype 5adenovirus (Ad5) in which human E2F-1 promoter drives expression of theessential E1a viral genes, thereby restricting viral replication andcytotoxicity to Rb pathway-defective tumor cells (Cold Genesys, Inc.)(see, e.g., Clinical Trial Identifier: NCT02143804); or

DNX-2401 (formerly named Delta-24-RGD), which is an adenovirus that hasbeen engineered to replicate selectively in retinoblastoma (Rb)-pathwaydeficient cells and to infect cells that express certain RGD-bindingintegrins more efficiently (Clinica Universidad de Navarra, Universidadde Navarra/DNAtrix, Inc.) (see, e.g., Clinical Trial Identifier:NCT01956734).

In some embodiments, an oncolytic virus described herein isadministering by injection, e.g., subcutaneous, intra-arterial,intravenous, intramuscular, intrathecal, or intraperitoneal injection.In embodiments, an oncolytic virus described herein is administeredintratumorally, transdermally, transmucosally, orally, intranasally, orvia pulmonary administration.

In an embodiment, cells expressing a CAR described herein areadministered to a subject in combination with a molecule that decreasesthe Treg cell population. Methods that decrease the number of (e.g.,deplete) Treg cells are known in the art and include, e.g., CD25depletion, cyclophosphamide administration, modulating GITR function.Without wishing to be bound by theory, it is believed that reducing thenumber of Treg cells in a subject prior to apheresis or prior toadministration of a CAR-expressing cell described herein reduces thenumber of unwanted immune cells (e.g., Tregs) in the tumormicroenvironment and reduces the subject's risk of relapse. In oneembodiment, a CAR expressing cell described herein are administered to asubject in combination with a molecule targeting GITR and/or modulatingGITR functions, such as a GITR agonist and/or a GITR antibody thatdepletes regulatory T cells (Tregs). In embodiments, cells expressing aCAR described herein are administered to a subject in combination withcyclophosphamide. In one embodiment, the GITR binding molecules and/ormolecules modulating GITR functions (e.g., GITR agonist and/or Tregdepleting GITR antibodies) are administered prior to administration ofthe CAR-expressing cell. For example, in one embodiment, the GITRagonist can be administered prior to apheresis of the cells. Inembodiments, cyclophosphamide is administered to the subject prior toadministration (e.g., infusion or re-infusion) of the CAR-expressingcell or prior to aphersis of the cells. In embodiments, cyclophosphamideand an anti-GITR antibody are administered to the subject prior toadministration (e.g., infusion or re-infusion) of the CAR-expressingcell or prior to apheresis of the cells. In one embodiment, the subjecthas cancer (e.g., a solid cancer or a hematological cancer such as ALLor CLL). In an embodiment, the subject has CLL. In embodiments, thesubject has ALL. In embodiments, the subject has a solid cancer, e.g., asolid cancer described herein. Exemplary GITR agonists include, e.g.,GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITRantibodies) such as, e.g., a GITR fusion protein described in U.S. Pat.No. 6,111,090, European Patent No.: 090505B1, U.S. Pat. No. 8,586,023,PCT Publication Nos.: WO 2010/003118 and 2011/090754, or an anti-GITRantibody described, e.g., in U.S. Pat. No. 7,025,962, European PatentNo.: 1947183B1, U.S. Pat. Nos. 7,812,135, 8,388,967, 8,591,886, EuropeanPatent No.: EP 1866339, PCT Publication No.: WO 2011/028683, PCTPublication No.: WO 2013/039954, PCT Publication No.: WO2005/007190, PCTPublication No.: WO 2007/133822, PCT Publication No.: WO2005/055808, PCTPublication No.: WO 99/40196, PCT Publication No.: WO 2001/03720, PCTPublication No.: WO99/20758, PCT Publication No.: WO2006/083289, PCTPublication No.: WO 2005/115451, U.S. Pat. No. 7,618,632, and PCTPublication No.: WO 2011/051726.

In one embodiment, a CAR expressing cell described herein isadministered to a subject in combination with an mTOR inhibitor, e.g.,an mTOR inhibitor described herein, e.g., a rapalog such as everolimus.In one embodiment, the mTOR inhibitor is administered prior to theCAR-expressing cell. For example, in one embodiment, the mTOR inhibitorcan be administered prior to apheresis of the cells.

In one embodiment, a CAR expressing cell described herein isadministered to a subject in combination with a GITR agonist, e.g., aGITR agonist described herein. In one embodiment, the GITR agonist isadministered prior to the CAR-expressing cell. For example, in oneembodiment, the GITR agonist can be administered prior to apheresis ofthe cells.

In one embodiment, a CAR expressing cell described herein isadministered to a subject in combination with a protein tyrosinephosphatase inhibitor, e.g., a protein tyrosine phosphatase inhibitordescribed herein. In one embodiment, the protein tyrosine phosphataseinhibitor is an SHP-1 inhibitor, e.g., an SHP-1 inhibitor describedherein, such as, e.g., sodium stibogluconate. In one embodiment, theprotein tyrosine phosphatase inhibitor is an SHP-2 inhibitor.

In one embodiment, a CAR-expressing cell described herein can be used incombination with a kinase inhibitor. In one embodiment, the kinaseinhibitor is a CDK4 inhibitor, e.g., a CDK4 inhibitor described herein,e.g., a CD4/6 inhibitor, such as, e.g.,6-Acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one,hydrochloride (also referred to as palbociclib or PD0332991). In oneembodiment, the kinase inhibitor is a BTK inhibitor, e.g., a BTKinhibitor described herein, such as, e.g., ibrutinib. In one embodiment,the kinase inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitordescribed herein, such as, e.g., rapamycin, a rapamycin analog, OSI-027.The mTOR inhibitor can be, e.g., an mTORC1 inhibitor and/or an mTORC2inhibitor, e.g., an mTORC1 inhibitor and/or mTORC2 inhibitor describedherein. In one embodiment, the kinase inhibitor is a MNK inhibitor,e.g., a MNK inhibitor described herein, such as, e.g.,4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d] pyrimidine. The MNKinhibitor can be, e.g., a MNK1a, MNK1b, MNK2a and/or MNK2b inhibitor. Inone embodiment, the kinase inhibitor is a dual PI3K/mTOR inhibitordescribed herein, such as, e.g., PF-04695102. In one embodiment, thekinase inhibitor is a DGK inhibitor, e.g., a DGK inhibitor describedherein, such as, e.g., DGKinh1 (D5919) or DGKinh2 (D5794).

In one embodiment, the kinase inhibitor is a CDK4 inhibitor selectedfrom aloisine A; flavopiridol or HMR-1275,2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone;crizotinib (PF-02341066;2-(2-Chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one,hydrochloride (P276-00);1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine(RAF265); indisulam (E7070); roscovitine (CYC202); palbociclib(PD0332991); dinaciclib (SCH727965);N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide(BMS 387032);4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoicacid (MLN8054);543-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine(AG-024322); 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidN-(piperidin-4-yl)amide (AT7519);4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine(AZD5438); and XL281 (BMS908662).

In one embodiment, the kinase inhibitor is a CDK4 inhibitor, e.g.,palbociclib (PD0332991), and the palbociclib is administered at a doseof about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g., 75 mg, 100 mg or 125mg) daily for a period of time, e.g., daily for 14-21 days of a 28 daycycle, or daily for 7-12 days of a 21 day cycle. In one embodiment, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of palbociclib areadministered.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a cyclin-dependent kinase (CDK) 4 or 6inhibitor, e.g., a CDK4 inhibitor or a CDK6 inhibitor described herein.In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a CDK4/6 inhibitor (e.g., an inhibitorthat targets both CDK4 and CDK6), e.g., a CDK4/6 inhibitor describedherein. In an embodiment, the subject has MCL. MCL is an aggressivecancer that is poorly responsive to currently available therapies, i.e.,essentially incurable. In many cases of MCL, cyclin D1 (a regulator ofCDK4/6) is expressed (e.g., due to chromosomal translocation involvingimmunoglobulin and Cyclin D1 genes) in MCL cells. Thus, without beingbound by theory, it is thought that MCL cells are highly sensitive toCDK4/6 inhibition with high specificity (i.e., minimal effect on normalimmune cells). CDK4/6 inhibitors alone have had some efficacy intreating MCL, but have only achieved partial remission with a highrelapse rate. An exemplary CDK4/6 inhibitor is LEE011 (also calledribociclib), the structure of which is shown below.

Without being bound by theory, it is believed that administration of aCAR-expressing cell described herein with a CDK4/6 inhibitor (e.g.,LEE011 or other CDK4/6 inhibitor described herein) can achieve higherresponsiveness, e.g., with higher remission rates and/or lower relapserates, e.g., compared to a CDK4/6 inhibitor alone.

In one embodiment, the kinase inhibitor is a BTK inhibitor selected fromibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224;CC-292; ONO-4059; CNX-774; and LFM-A13. In a preferred embodiment, theBTK inhibitor does not reduce or inhibit the kinase activity ofinterleukin-2-inducible kinase (ITK), and is selected from GDC-0834;RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; andLFM-A13.

In one embodiment, the kinase inhibitor is a BTK inhibitor, e.g.,ibrutinib (PCI-32765). In embodiments, a CAR-expressing cell describedherein is administered to a subject in combination with a BTK inhibitor(e.g., ibrutinib). In embodiments, a CAR-expressing cell describedherein is administered to a subject in combination with ibrutinib (alsocalled PCI-32765). The structure of ibrutinib(1-[(3R)-344-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one)is shown below.

In embodiments, the subject has CLL, mantle cell lymphoma (MCL), orsmall lymphocytic lymphoma (SLL). For example, the subject has adeletion in the short arm of chromosome 17 (del(17p), e.g., in aleukemic cell). In other examples, the subject does not have a del(17p).In embodiments, the subject has relapsed CLL or SLL, e.g., the subjecthas previously been administered a cancer therapy (e.g., previously beenadministered one, two, three, or four prior cancer therapies). Inembodiments, the subject has refractory CLL or SLL. In otherembodiments, the subject has follicular lymphoma, e.g., relapse orrefractory follicular lymphoma. In some embodiments, ibrutinib isadministered at a dosage of about 300-600 mg/day (e.g., about 300-350,350-400, 400-450, 450-500, 500-550, or 550-600 mg/day, e.g., about 420mg/day or about 560 mg/day), e.g., orally. In embodiments, the ibrutinibis administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600mg (e.g., 250 mg, 420 mg or 560 mg) daily for a period of time, e.g.,daily for 21 day cycle cycle, or daily for 28 day cycle. In oneembodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles ofibrutinib are administered. In some embodiments, ibrutinib isadministered in combination with rituximab. See, e.g., Burger et al.(2013) Ibrutinib In Combination With Rituximab (iR) Is Well Toleratedand Induces a High Rate Of Durable Remissions In Patients With High-RiskChronic Lymphocytic Leukemia (CLL): New, Updated Results Of a Phase IITrial In 40 Patients, Abstract 675 presented at 55^(th) ASH AnnualMeeting and Exposition, New Orleans, La. 7-10 December Without beingbound by theory, it is thought that the addition of ibrutinib enhancesthe T cell proliferative response and may shift T cells from aT-helper-2 (Th2) to T-helper-1 (Th1) phenotype. Th1 and Th2 arephenotypes of helper T cells, with Th1 versus Th2 directing differentimmune response pathways. A Th1 phenotype is associated withproinflammatory responses, e.g., for killing cells, such asintracellular pathogens/viruses or cancerous cells, or perpetuatingautoimmune responses. A Th2 phenotype is associated with eosinophilaccumulation and anti-inflammatory responses.

In some embodiments of the methods, uses, and compositions herein, theBTK inhibitor is a BTK inhibitor described in International ApplicationWO/2015/079417, which is herein incorporated by reference in itsentirety. For instance, in some embodiments, the BTK inhibitor is acompound of formula (I) or a pharmaceutically acceptable salt thereof;

wherein,

R1 is hydrogen, C1-C6 alkyl optionally substituted by hydroxy;

R2 is hydrogen or halogen;

R3 is hydrogen or halogen;

R4 is hydrogen;

R5 is hydrogen or halogen;

or R4 and R5 are attached to each other and stand for a bond, —CH2-,—CH2-CH2-, —CH═CH—, —CH═CH—CH2-; —CH2-CH═CH—; or —CH2-CH2—CH2-;

R6 and R7 stand independently from each other for H, C1-C6 alkyloptionally substituted by hydroxyl, C3-C6 cycloalkyl optionallysubstituted by halogen or hydroxy, or halogen;

R8, R9, R, R′, R10 and R11 independently from each other stand for H, orC1-C6 alkyl optionally substituted by C1-C6 alkoxy; or any two of R8,R9, R, R′, R10 and R11 together with the carbon atom to which they arebound may form a 3-6 membered saturated carbocyclic ring;

R12 is hydrogen or C1-C6 alkyl optionally substituted by halogen orC1-C6 alkoxy;

or R12 and any one of R8, R9, R, R′, R10 or R11 together with the atomsto which they are bound may form a 4, 5, 6 or 7 membered azacyclic ring,which ring may optionally be substituted by halogen, cyano, hydroxyl,C1-C6 alkyl or C1-C6 alkoxy;

n is 0 or 1; and

R13 is C2-C6 alkenyl optionally substituted by C1-C6 alkyl, C1-C6 alkoxyor N,N-di-C1-C6 alkyl amino; C2-C6 alkynyl optionally substituted byC1-C6 alkyl or C1-C6 alkoxy; or C2-C6 alkylenyl oxide optionallysubstituted by C1-C6 alkyl.

In some embodiments, the BTK inhibitor of Formula I is chosen from:N-(3-(5-((1-Acryloylazetidin-3-yl)oxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(E)-N-(3-(6-Amino-5-((1-(but-2-enoyl)azetidin-3-yl)oxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5((1-propioloylazetidin-3-yl)oxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-((1-(but-2-ynoyl)azetidin-3-yl)oxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(5-((1-Acryloylpiperidin-4-yl)oxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(2-(N-methylacrylamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(E)-N-(3-(6-Amino-5-(2-(N-methylbut-2-enamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(2-(N-methylpropiolamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(E)-N-(3-(6-Amino-5-(2-(4-methoxy-N-methylbut-2-enamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(2-(N-methylbut-2-ynamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(2-((4-Amino-6-(3-(4-cyclopropyl-2-fluorobenzamido)-5-fluoro-2-methylphenyl)pyrimidin-5-yl)oxy)ethyl)-N-methyloxirane-2-carboxamide;N-(2-((4-Amino-6-(3-(6-cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)phenyl)pyrimidin-5-yl)oxy)ethyl)-N-methylacrylamide;N-(3-(5-(2-Acrylamidoethoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(2-(N-ethylacrylamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(2-(N-(2-fluoroethyl)acrylamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(5-((1-Acrylamidocyclopropyl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)—N-(3-(5-(2-Acrylamidopropoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)—N-(3-(6-Amino-5-(2-(but-2-ynamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)—N-(3-(6-Amino-5-(2-(N-methylacrylamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)—N-(3-(6-Amino-5-(2-(N-methylbut-2-ynamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(3-(N-methylacrylamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)—N-(3-(5-((1-Acryloylpyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)—N-(3-(6-Amino-5-((1-(but-2-ynoyl)pyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)-2-(3-(5-((1-Acryloylpyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6-cyclopropyl-3,4-dihydroisoquinolin-1(2H)-one;N-(2-((4-Amino-6-(3-(6-cyclopropyl-1-oxo-3,4-dihydroisoquinolin-2(1H)-yl)-5-fluoro-2-(hydroxymethyl)phenyl)pyrimidin-5-yl)oxy)ethyl)-N-methylacrylamide;N-(3-(5-(((2S,4R)-1-Acryloyl-4-methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(((2S,4R)-1-(but-2-ynoyl)-4-methoxypyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;2-(3-(5-(((2S,4R)-1-Acryloyl-4-methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6-cyclopropyl-3,4-dihydroisoquinolin-1(2H)-one;N-(3-(5-(((2S,4S)-1-Acryloyl-4-methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-54(2S,4S)-1-(but-2-ynoyl)-4-methoxypyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(5-((2S,4R)-1-Acryloyl-4-fluoropyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(6-Amino-5-(((2S,4R)-1-(but-2-ynoyl)-4-fluoropyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)—N-(3-(5-((1-Acryloylazetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)—N-(3-(6-Amino-5-((1-propioloylazetidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(S)-2-(3-(5-((1-Acryloylazetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6-cyclopropyl-3,4-dihydroisoquinolin-1(2H)-one;(R)—N-(3-(5-((1-Acryloylazetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;(R)—N-(3-(5-((l-Acryloylpiperidin-3-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(5-(((2R,3S)-1-Acryloyl-3-methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;N-(3-(5-(((2S,4R)-1-Acryloyl-4-cyanopyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;orN-(3-(5-(((2S,4S)-1-Acryloyl-4-cyanopyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide.

Unless otherwise provided, the chemical terms used above in describingthe BTK inhibitor of Formula I are used according to their meanings asset out in International Application WO/2015/079417, which is hereinincorporated by reference in its entirety.

In one embodiment, the kinase inhibitor is an mTOR inhibitor selectedfrom temsirolimus; ridaforolimus (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669; everolimus(RAD001); rapamycin (AY22989); simapimod;(542,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502); andN²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-a-aspartylL-serine-(SEQID NO: 378), inner salt (SF1126); and XL765.

In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g.,rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg) daily for a periodof time, e.g., daily for 21 day cycle cycle, or daily for 28 day cycle.In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cyclesof rapamycin are administered. In one embodiment, the kinase inhibitoris an mTOR inhibitor, e.g., everolimus and the everolimus isadministered at a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (e.g., 10 mg)daily for a period of time, e.g., daily for 28 day cycle. In oneembodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles ofeverolimus are administered.

In one embodiment, the kinase inhibitor is an MNK inhibitor selectedfrom CGP052088; 4-amino-3-(p-fluorophenylamino)-pyrazolo [3,4-d]pyrimidine (CGP57380); cercosporamide; ETC-1780445-2; and4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d] pyrimidine.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a phosphoinositide 3-kinase (PI3K)inhibitor (e.g., a PI3K inhibitor described herein, e.g., idelalisib orduvelisib) and/or rituximab. In embodiments, a CAR-expressing celldescribed herein is administered to a subject in combination withidelalisib and rituximab. In embodiments, a CAR-expressing celldescribed herein is administered to a subject in combination withduvelisib and rituximab. Idelalisib (also called GS-1101 or CAL-101;Gilead) is a small molecule that blocks the delta isoform of PI3K. Thestructure of idelalisib(5-Fluoro-3-phenyl-2-[(15)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone)is shown below.

Duvelisib (also called IPI-145; Infinity Pharmaceuticals and Abbvie) isa small molecule that blocks PI3K-δ,γ. The structure of duvelisib(8-Chloro-2-phenyl-3-[(1S)-1-(9H-purin-6-ylamino)ethyl]-1(2H)-isoquinolinone)is shown below.

In embodiments, the subject has CLL. In embodiments, the subject hasrelapsed CLL, e.g., the subject has previously been administered acancer therapy (e.g., previously been administered an anti-CD20 antibodyor previously been administered ibrutinib). For example, the subject hasa deletion in the short arm of chromosome 17 (del(17p), e.g., in aleukemic cell). In other examples, the subject does not have a del(17p).In embodiments, the subject comprises a leukemic cell comprising amutation in the immunoglobulin heavy-chain variable-region (IgV_(H))gene. In other embodiments, the subject does not comprise a leukemiccell comprising a mutation in the immunoglobulin heavy-chainvariable-region (IgV_(H)) gene. In embodiments, the subject has adeletion in the long arm of chromosome 11 (del(11q)). In otherembodiments, the subject does not have a del(11q). In embodiments,idelalisib is administered at a dosage of about 100-400 mg (e.g.,100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, 275-300,325-350, 350-375, or 375-400 mg), e.g., BID. In embodiments, duvelisibis administered at a dosage of about 15-100 mg (e.g., about 15-25,25-50, 50-75, or 75-100 mg), e.g., twice a day. In embodiments,rituximab is administered at a dosage of about 350-550 mg/m² (e.g.,350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m²), e.g.,intravenously.

In one embodiment, the kinase inhibitor is a dual phosphatidylinositol3-kinase (PI3K) and mTOR inhibitor selected from2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF-04691502);N-[4-[4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl)phenyl]urea(PF-05212384, PKI-587);2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile(BEZ-235); apitolisib (GDC-0980, RG7422);2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide(GSK2126458);8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-oneMaleic acid (NVP-BGT226);3-[4-(4-Morpholinylpyrido[3,2;4,5]furo[3,2-d]pyrimidin-2-yl]phenol(PI-103);5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine(VS-5584, SB2343); andN42-[(3,5-Dimethoxyphenyl)amino]quinoxalin-3-yl]-4-[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide(XL765).

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with an anaplastic lymphoma kinase (ALK)inhibitor. Exemplary ALK kinases include but are not limited tocrizotinib (Pfizer), ceritinib (Novartis), alectinib (Chugai),brigatinib (also called AP26113; Ariad), entrectinib (Ignyta),PF-06463922 (Pfizer), TSR-011 (Tesaro) (see, e.g., Clinical TrialIdentifier No. NCT02048488), CEP-37440 (Teva), and X-396 (Xcovery). Insome embodiments, the subject has a solid cancer, e.g., a solid cancerdescribed herein, e.g., lung cancer.

The chemical name of crizotinib is3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine.The chemical name of ceritinib is5-Chloro-N²-[2-isopropoxy-5-methyl-4-(4-piperidinyl)phenyl]-N⁴-[2-(isopropylsulfonyl)phenyl]-2,4-pyrimidinediamine.The chemical name of alectinib is9-ethyl-6,6-dimethyl-8-(4-morpholinopiperidin-1-yl)-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile.The chemical name of brigatinib is5-Chloro-N²-{4-[4-(dimethylamino)-1-piperidinyl]-2-methoxyphenyl}-N⁴-[2-(dimethylphosphoryl)phenyl]-2,4-pyrimidinediamine.The chemical name of entrectinib isN-(5-(3,5-difluorobenzyl)-1H-indazol-3-yl)-4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-4-yl)amino)benzamide.The chemical name of PF-06463922 is(10R)-7-Amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]-benzoxadiazacyclotetradecine-3-carbonitrile.The chemical structure of CEP-37440 is(S)-2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide.The chemical name of X-396 is(R)-6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-N-(4-(4-methylpiperazine-1-carbonyl)phenyl)pyridazine-3-carboxamide.

Drugs that inhibit either the calcium dependent phosphatase calcineurin(cyclosporine and FK506) or inhibit the p70S6 kinase that is importantfor growth factor induced signaling (rapamycin). (Liu et al., Cell66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer etal., Curr. Opin. Immun. 5:763-773, 1993) can also be used. In a furtheraspect, the cell compositions of the present invention may beadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, and/orantibodies such as OKT3 or CAMPATH. In one aspect, the cell compositionsof the present invention are administered following B-cell ablativetherapy such as agents that react with CD20, e.g., Rituxan. For example,in one embodiment, subjects may undergo standard treatment with highdose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant,subjects receive an infusion of the expanded immune cells of the presentinvention. In an additional embodiment, expanded cells are administeredbefore or following surgery.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with an indoleamine 2,3-dioxygenase (IDO)inhibitor. IDO is an enzyme that catalyzes the degradation of the aminoacid, L-tryptophan, to kynurenine. Many cancers overexpress IDO, e.g.,prostatic, colorectal, pancreatic, cervical, gastric, ovarian, head, andlung cancer. pDCs, macrophages, and dendritic cells (DCs) can expressIDO. Without being bound by theory, it is thought that a decrease inL-tryptophan (e.g., catalyzed by IDO) results in an immunosuppressivemilieu by inducing T-cell anergy and apoptosis. Thus, without beingbound by theory, it is thought that an IDO inhibitor can enhance theefficacy of a CAR-expressing cell described herein, e.g., by decreasingthe suppression or death of a CAR-expressing immune cell. Inembodiments, the subject has a solid tumor, e.g., a solid tumordescribed herein, e.g., prostatic, colorectal, pancreatic, cervical,gastric, ovarian, head, or lung cancer. Exemplary inhibitors of IDOinclude but are not limited to 1-methyl-tryptophan, indoximod (NewLinkGenetics) (see, e.g., Clinical Trial Identifier Nos. NCT01191216;NCT01792050), and INCB024360 (Incyte Corp.) (see, e.g., Clinical TrialIdentifier Nos. NCT01604889; NCT01685255)

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a modulator of myeloid-derivedsuppressor cells (MDSCs). MDSCs accumulate in the periphery and at thetumor site of many solid tumors. These cells suppress T cell responses,thereby hindering the efficacy of CAR-expressing cell therapy. Withoutbeing bound by theory, it is thought that administration of a MDSCmodulator enhances the efficacy of a CAR-expressing cell describedherein. In an embodiment, the subject has a solid tumor, e.g., a solidtumor described herein, e.g., glioblastoma. Exemplary modulators ofMDSCs include but are not limited to MCS110 and BLZ945. MCS110 is amonoclonal antibody (mAb) against macrophage colony-stimulating factor(M-CSF). See, e.g., Clinical Trial Identifier No. NCT00757757. BLZ945 isa small molecule inhibitor of colony stimulating factor 1 receptor(CSF1R). See, e.g., Pyonteck et al. Nat. Med. 19(2013):1264-72. Thestructure of BLZ945 is shown below.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a CD19 CART cell (e.g., CTL019, e.g.,as described in WO2012/079000, incorporated herein by reference). Inembodiments, the subject has acute myeloid leukemia (AML), e.g., a CD19positive AML, or a CD19 negative AML. In embodiments, the subject has aCD19+ lymphoma, e.g., a CD19+ Non-Hodgkin's Lymphoma (NHL), a CD19+FL,or a CD19+ DLBCL. In embodiments, the subject has a relapsed orrefractory CD19+ lymphoma. In embodiments, a lymphodepletingchemotherapy is administered to the subject prior to, concurrently with,or after administration (e.g., infusion) of CD19 CART cells. In anexample, the lymphodepleting chemotherapy is administered to the subjectprior to administration of CD19 CART cells. For example, thelymphodepleting chemotherapy ends 1-4 days (e.g., 1, 2, 3, or 4 days)prior to CD19 CART cell infusion. In embodiments, multiple doses of CD19CART cells are administered, e.g., as described herein. For example, asingle dose comprises about 5×10⁸ CD19 CART cells. In embodiments, alymphodepleting chemotherapy is administered to the subject prior to,concurrently with, or after administration (e.g., infusion) of aCAR-expressing cell described herein, e.g., a non-CD19 CAR-expressingcell. In embodiments, a CD19 CART is administered to the subject priorto, concurrently with, or after administration (e.g., infusion) of anon-CD19 CAR-expressing cell, e.g., a non-CD19 CAR-expressing celldescribed herein.

In some embodiments, a CAR-expressing cell described herein isadministered to a subject in combination with a CD19 CAR-expressingcell, e.g., CTL019, e.g., as described in WO2012/079000, incorporatedherein by reference, for treatment of a disease associated with theexpression of CD33, e.g., a cancer described herein. Without being boundby theory, it is believed that administering a CD19 CAR-expressing cellin combination with a CAR-expressing cell improves the efficacy of aCAR-expressing cell described herein by targeting early lineage cancercells, e.g., cancer stem cells, modulating the immune response,depleting regulatory B cells, and/or improving the tumormicroenvironment. For example, a CD19 CAR-expressing cell targets cancercells that express early lineage markers, e.g., cancer stem cells andCD19-expressing cells, while the CAR-expressing cell described hereintargets cancer cells that express later lineage markers, e.g., CD33.This preconditioning approach can improve the efficacy of theCAR-expressing cell described herein. In such embodiments, the CD19CAR-expressing cell is administered prior to, concurrently with, orafter administration (e.g., infusion) of a CAR-expressing cell describedherein.

In embodiments, a CAR-expressing cell described herein also expresses aCAR targeting CD19, e.g., a CD19 CAR. In an embodiment, the cellexpressing a CAR described herein and a CD19 CAR is administered to asubject for treatment of a cancer described herein, e.g., AML. In anembodiment, the configurations of one or both of the CAR moleculescomprise a primary intracellular signaling domain and a costimulatorysignaling domain. In another embodiment, the configurations of one orboth of the CAR molecules comprise a primary intracellular signalingdomain and two or more, e.g., 2, 3, 4, or 5 or more, costimulatorysignaling domains. In such embodiments, the CAR molecule describedherein and the CD19 CAR may have the same or a different primaryintracellular signaling domain, the same or different costimulatorysignaling domains, or the same number or a different number ofcostimulatory signaling domains. Alternatively, the CAR described hereinand the CD19 CAR are configured as a split CAR, in which one of the CARmolecules comprises an antigen binding domain and a costimulatory domain(e.g., 4-1BB), while the other CAR molecule comprises an antigen bindingdomain and a primary intracellular signaling domain (e.g., CD3 zeta).

In some embodiments, a CAR-expressing cell described herein isadministered to a subject in combination with a interleukin-15 (IL-15)polypeptide, a interleukin-15 receptor alpha (IL-15Ra) polypeptide, or acombination of both a IL-15 polypeptide and a IL-15Ra polypeptide e.g.,hetIL-15 (Admune Therapeutics, LLC). hetIL-15 is a heterodimericnon-covalent complex of IL-15 and IL-15Ra. hetIL-15 is described in,e.g., U.S. Pat. No. 8,124,084, U.S. 2012/0177598, U.S. 2009/0082299,U.S. 2012/0141413, and U.S. 2011/0081311, incorporated herein byreference. In embodiments, het-IL-15 is administered subcutaneously. Inembodiments, the subject has a cancer, e.g., solid cancer, e.g.,melanoma or colon cancer. In embodiments, the subject has a metastaticcancer.

In embodiments, a subject having a disease described herein, e.g., ahematological disorder, e.g., AML or MDS, is administered aCAR-expressing cell described herein in combination with an agent, e.g.,cytotoxic or chemotherapy agent, a biologic therapy (e.g., antibody,e.g., monoclonal antibody, or cellular therapy), or an inhibitor (e.g.,kinase inhibitor). In embodiments, the subject is administered aCAR-expressing cell described herein in combination with a cytotoxicagent, e.g., CPX-351 (Celator Pharmaceuticals), cytarabine,daunorubicin, vosaroxin (Sunesis Pharmaceuticals), sapacitabine(Cyclacel Pharmaceuticals), idarubicin, or mitoxantrone. CPX-351 is aliposomal formulation comprising cytarabine and daunorubicin at a 5:1molar ratio. In embodiments, the subject is administered aCAR-expressing cell described herein in combination with ahypomethylating agent, e.g., a DNA methyltransferase inhibitor, e.g.,azacitidine or decitabine. In embodiments, the subject is administered aCAR-expressing cell described herein in combination with a biologictherapy, e.g., an antibody or cellular therapy, e.g., 225Ac-lintuzumab(Actimab-A; Actinium Pharmaceuticals), IPH2102 (Innate Pharma/BristolMyers Squibb), SGN-CD33A (Seattle Genetics), or gemtuzumab ozogamicin(Mylotarg; Pfizer). SGN-CD33A is an antibody-drug conjugate (ADC)comprising a pyrrolobenzodiazepine dimer that is attached to ananti-CD33 antibody. Actimab-A is an anti-CD33 antibody (lintuzumab)labeled with actinium. IPH2102 is a monoclonal antibody that targetskiller immunoglobulin-like receptors (KIRs). In embodiments, the subjectis administered a CAR-expressing cell described herein in combination aFLT3 inhibitor, e.g., sorafenib (Bayer), midostaurin (Novartis),quizartinib (Daiichi Sankyo), crenolanib (Arog Pharmaceuticals), PLX3397(Daiichi Sankyo), AKN-028 (Akinion Pharmaceuticals), or ASP2215(Astellas). In embodiments, the subject is administered a CAR-expressingcell described herein in combination with an isocitrate dehydrogenase(IDH) inhibitor, e.g., AG-221 (Celgene/Agios) or AG-120 (Agios/Celgene).In embodiments, the subject is administered a CAR-expressing celldescribed herein in combination with a cell cycle regulator, e.g.,inhibitor of polo-like kinase 1 (Plk1), e.g., volasertib (BoehringerIngelheim); or an inhibitor of cyclin-dependent kinase 9 (Cdk9), e.g.,alvocidib (Tolero Pharmaceuticals/Sanofi Aventis). In embodiments, thesubject is administered a CAR-expressing cell described herein incombination with a B cell receptor signaling network inhibitor, e.g., aninhibitor of B-cell lymphoma 2 (Bcl-2), e.g., venetoclax (Abbvie/Roche);or an inhibitor of Bruton's tyrosine kinase (Btk), e.g., ibrutinib(Pharmacyclics/Johnson & Johnson Janssen Pharmaceutical). Inembodiments, the subject is administered a CAR-expressing cell describedherein in combination with an inhibitor of M1 aminopeptidase, e.g.,tosedostat (CTI BioPharma/Vernalis); an inhibitor of histone deacetylase(HDAC), e.g., pracinostat (MEI Pharma); a multi-kinase inhibitor, e.g.,rigosertib (Onconova Therapeutics/Baxter/SymBio); or a peptidic CXCR4inverse agonist, e.g., BL-8040 (BioLineRx). In embodiments, the subjectis administered a CD33-targeting CAR-expressing cell in combination witha CAR-expressing cell that targets an antigen other than CD33, e.g.,CLL, BCMA, CD123, CD19, FLT-3, or folate receptor beta.

In another embodiment, the subjects receive an infusion of the CART33cell compositions of the present invention prior to transplantation,e.g., allogeneic stem cell transplant, of cells. In a preferredembodiment, the CART33 cells transiently express CAR33, e.g., byelectroporation of an mRNA anti-CD33 CAR, whereby the expression of theCAR33 is terminated prior to infusion of donor stem cells to avoidengraftment failure.

Some patients may experience allergic reactions to the compounds of thepresent invention and/or other anti-cancer agent(s) during or afteradministration; therefore, anti-allergic agents are often administeredto minimize the risk of an allergic reaction. Suitable anti-allergicagents include corticosteroids, such as dexamethasone (e.g., Decadron®),beclomethasone (e.g., Beclovent®), hydrocortisone (also known ascortisone, hydrocortisone sodium succinate, hydrocortisone sodiumphosphate, and sold under the tradenames Ala-Cort®, hydrocortisonephosphate, Solu-Cortef®, Hydrocort Acetate® and Lanacort®), prednisolone(sold under the tradenames Delta-Cortel®, Orapred®, Pediapred® andPrelone®), prednisone (sold under the tradenames Deltasone®, LiquidRed®, Meticorten® and Orasone®), methylprednisolone (also known as6-methylprednisolone, methylprednisolone acetate, methylprednisolonesodium succinate, sold under the tradenames Duralone®, Medralone®,Medrol®, M-Prednisol® and Solu-Medrol®); antihistamines, such asdiphenhydramine (e.g., Benadryl®), hydroxyzine, and cyproheptadine; andbronchodilators, such as the beta-adrenergic receptor agonists,albuterol (e.g., Proventil®), and terbutaline (Brethine®).

Some patients may experience nausea during and after administration ofthe compound of the present invention and/or other anti-cancer agent(s);therefore, anti-emetics are used in preventing nausea (upper stomach)and vomiting. Suitable anti-emetics include aprepitant (Emend®),ondansetron (Zofran®), granisetron HCl (Kytril®), lorazepam (Ativan®.dexamethasone (Decadron®), prochlorperazine (Compazine®), casopitant(Rezonic® and Zunrisa®), and combinations thereof.

Medication to alleviate the pain experienced during the treatment periodis often prescribed to make the patient more comfortable. Commonover-the-counter analgesics, such Tylenol®, are often used. However,opioid analgesic drugs such as hydrocodone/paracetamol orhydrocodone/acetaminophen (e.g., Vicodin®), morphine (e.g., Astramorph®or Avinza®), oxycodone (e.g., OxyContin® or Percocet®), oxymorphonehydrochloride (Opana®), and fentanyl (e.g., Duragesic®) are also usefulfor moderate or severe pain.

In an effort to protect normal cells from treatment toxicity and tolimit organ toxicities, cytoprotective agents (such as neuroprotectants,free-radical scavengers, cardioprotectors, anthracycline extravasationneutralizers, nutrients and the like) may be used as an adjunct therapy.Suitable cytoprotective agents include Amifostine (Ethyol®), glutamine,dimesna (Tavocept®), mesna (Mesnex®), dexrazoxane (Zinecard® orTotect®), xaliproden (Xaprila®), and leucovorin (also known as calciumleucovorin, citrovorum factor and folinic acid).

The structure of the active compounds identified by code numbers,generic or trade names may be taken from the actual edition of thestandard compendium “The Merck Index” or from databases, e.g. PatentsInternational (e.g. IMS World Publications).

The above-mentioned compounds, which can be used in combination with acompound of the present invention, can be prepared and administered asdescribed in the art, such as in the documents cited above.

In one embodiment, the present invention provides pharmaceuticalcompositions comprising at least one compound of the present invention(e.g., a compound of the present invention) or a pharmaceuticallyacceptable salt thereof together with a pharmaceutically acceptablecarrier suitable for administration to a human or animal subject, eitheralone or together with other anti-cancer agents.

In one embodiment, the present invention provides methods of treatinghuman or animal subjects suffering from a cellular proliferativedisease, such as cancer. The present invention provides methods oftreating a human or animal subject in need of such treatment, comprisingadministering to the subject a therapeutically effective amount of acompound of the present invention (e.g., a compound of the presentinvention) or a pharmaceutically acceptable salt thereof, either aloneor in combination with other anti-cancer agents.

In particular, compositions will either be formulated together as acombination therapeutic or administered separately.

In combination therapy, the compound of the present invention and otheranti-cancer agent(s) may be administered either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the twocompounds in the body of the patient.

In a preferred embodiment, the compound of the present invention and theother anti-cancer agent(s) is generally administered sequentially in anyorder by infusion or orally. The dosing regimen may vary depending uponthe stage of the disease, physical fitness of the patient, safetyprofiles of the individual drugs, and tolerance of the individual drugs,as well as other criteria well-known to the attending physician andmedical practitioner(s) administering the combination. The compound ofthe present invention and other anti-cancer agent(s) may be administeredwithin minutes of each other, hours, days, or even weeks apart dependingupon the particular cycle being used for treatment. In addition, thecycle could include administration of one drug more often than the otherduring the treatment cycle and at different doses per administration ofthe drug.

In another aspect of the present invention, kits that include one ormore compound of the present invention and a combination partner asdisclosed herein are provided. Representative kits include (a) acompound of the present invention or a pharmaceutically acceptable saltthereof, (b) at least one combination partner, e.g., as indicated above,whereby such kit may comprise a package insert or other labelingincluding directions for administration.

A compound of the present invention may also be used to advantage incombination with known therapeutic processes, for example, theadministration of hormones or especially radiation. A compound of thepresent invention may in particular be used as a radiosensitizer,especially for the treatment of tumors which exhibit poor sensitivity toradiotherapy.

In one embodiment, the subject can be administered an agent whichreduces or ameliorates a side effect associated with the administrationof a CAR-expressing cell. Side effects associated with theadministration of a CAR-expressing cell include, but are not limited toCRS, and hemophagocytic lymphohistiocytosis (HLH), also termedMacrophage Activation Syndrome (MAS). Symptoms of CRS include highfevers, nausea, transient hypotension, hypoxia, and the like. CRS mayinclude clinical constitutional signs and symptoms such as fever,fatigue, anorexia, myalgias, arthalgias, nausea, vomiting, and headache.CRS may include clinical skin signs and symptoms such as rash. CRS mayinclude clinical gastrointestinal signs and symsptoms such as nausea,vomiting and diarrhea. CRS may include clinical respiratory signs andsymptoms such as tachypnea and hypoxemia. CRS may include clinicalcardiovascular signs and symptoms such as tachycardia, widened pulsepressure, hypotension, increased cardiac output (early) and potentiallydiminished cardiac output (late).

CRS may include clinical coagulation signs and symptoms such as elevatedd-dimer, hyperfibrinogenemia with or without bleeding. CRS may includeclinical renal signs and symptoms such as azotemia. CRS may includeclinical hepatic signs and symptoms such as transaminitis andhyperbilirubinemia. CRS may include clinical neurologic signs andsymptoms such as headache, mental status changes, confusion, delirium,word finding difficulty or frank aphasia, hallucinations, tremor,dymetria, altered gait, and seizures.

Accordingly, the methods described herein can comprise administering aCAR-expressing cell described herein to a subject and furtheradministering one or more agents to manage elevated levels of a solublefactor resulting from treatment with a CAR-expressing cell. In oneembodiment, the soluble factor elevated in the subject is one or more ofIFN-γ, TNFα, IL-2 and IL-6. In an embodiment, the factor elevated in thesubject is one or more of IL-1, GM-CSF, IL-10, IL-8, IL-5 andfraktalkine. Therefore, an agent administered to treat this side effectcan be an agent that neutralizes one or more of these soluble factors.In one embodiment, the agent that neutralizes one or more of thesesoluble forms is an antibody or antibody fragment. Examples of suchagents include, but are not limited to a steroid (e.g., corticosteroid),an inhibitor of TNFα, and an inhibitor of IL-6. An example of a TNFαinhibitor is an anti-TNFα antibody molecule such as, infliximab,adalimumab, certolizumab pegol, and golimumab. Another example of a TNFαinhibitor is a fusion protein such as entanercept. Small moleculeinhibitor of TNFα include, but are not limited to, xanthine derivatives(e.g. pentoxifylline) and bupropion. An example of an IL-6 inhibitor isan anti-IL-6 antibody molecule such as tocilizumab (toc), sarilumab,elsilimomab, CNTO 328, ALD518/BMS-945429, CNTO 136, CPSI-2364, CDP6038,VX30, ARGX-109, FE301, and FM101. In one embodiment, the anti-IL-6antibody molecule is tocilizumab. An example of an IL-1R based inhibitoris anakinra.

In some embodiment, the subject is administered a corticosteroid, suchas, e.g., methylprednisolone, hydrocortisone, among others.

In some embodiments, the subject is administered a vasopressor, such as,e.g., norepinephrine, dopamine, phenylephrine, epinephrine, vasopressin,or a combination thereof.

In an embodiment, the subject can be administered an antipyretic agent.In an embodiment, the subject can be administered an analgesic agent.

In one embodiment, the subject can be administered an agent whichenhances the activity of a CAR-expressing cell. For example, in oneembodiment, the agent can be an agent which inhibits an inhibitorymolecule, e.g., the agent is a checkpoint inhibitor. Inhibitorymolecules, e.g., Programmed Death 1 (PD1), can, in some embodiments,decrease the ability of a CAR-expressing cell to mount an immuneeffector response. Examples of inhibitory molecules include PD1, PD-L1,PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5),LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276),B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHCclass II, GAL9, adenosine, and TGFR beta. Inhibition of an inhibitorymolecule, e.g., by inhibition at the DNA, RNA or protein level, canoptimize a CAR-expressing cell performance. In embodiments, aninhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., adsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced shortpalindromic repeats (CRISPR), a transcription-activator like effectornuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., asdescribed herein, can be used to inhibit expression of an inhibitorymolecule in the CAR-expressing cell. In an embodiment the inhibitor isan shRNA. In an embodiment, the inhibitory molecule is inhibited withina CAR-expressing cell. In these embodiments, a dsRNA molecule thatinhibits expression of the inhibitory molecule is linked to the nucleicacid that encodes a component, e.g., all of the components, of the CAR.

In an embodiment, a nucleic acid molecule that encodes a dsRNA moleculethat inhibits expression of the molecule that modulates or regulates,e.g., inhibits, T-cell function is operably linked to a promoter, e.g.,a H1- or a U6-derived promoter such that the dsRNA molecule thatinhibits expression of the molecule that modulates or regulates, e.g.,inhibits, T-cell function is expressed, e.g., is expressed within aCAR-expressing cell. See e.g., Tiscornia G., “Development of LentiviralVectors Expressing siRNA,” Chapter 3, in Gene Transfer: Delivery andExpression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA, 2007; Brummelkamp T R,et al. (2002) Science 296: 550-553; Miyagishi M, et al. (2002) Nat.Biotechnol. 19: 497-500. In an embodiment the nucleic acid molecule thatencodes a dsRNA molecule that inhibits expression of the molecule thatmodulates or regulates, e.g., inhibits, T-cell function is present onthe same vector, e.g., a lentiviral vector, that comprises a nucleicacid molecule that encodes a component, e.g., all of the components, ofthe CAR. In such an embodiment, the nucleic acid molecule that encodes adsRNA molecule that inhibits expression of the molecule that modulatesor regulates, e.g., inhibits, T-cell function is located on the vector,e.g., the lentiviral vector, 5′- or 3′- to the nucleic acid that encodesa component, e.g., all of the components, of the CAR. The nucleic acidmolecule that encodes a dsRNA molecule that inhibits expression of themolecule that modulates or regulates, e.g., inhibits, T-cell functioncan be transcribed in the same or different direction as the nucleicacid that encodes a component, e.g., all of the components, of the CAR.In an embodiment the nucleic acid molecule that encodes a dsRNA moleculethat inhibits expression of the molecule that modulates or regulates,e.g., inhibits, T-cell function is present on a vector other than thevector that comprises a nucleic acid molecule that encodes a component,e.g., all of the components, of the CAR. In an embodiment, the nucleicacid molecule that encodes a dsRNA molecule that inhibits expression ofthe molecule that modulates or regulates, e.g., inhibits, T-cellfunction it transiently expressed within a CAR-expressing cell. In anembodiment, the nucleic acid molecule that encodes a dsRNA molecule thatinhibits expression of the molecule that modulates or regulates, e.g.,inhibits, T-cell function is stably integrated into the genome of aCAR-expressing cell. FIGS. 52A-52E depicts examples of vectors forexpressing a component, e.g., all of the components, of the CAR with adsRNA molecule that inhibits expression of the molecule that modulatesor regulates, e.g., inhibits, T-cell function.

Examples of dsRNA molecules useful for inhibiting expression of amolecule that modulates or regulates, e.g., inhibits, T-cell function,wherein the molecule that modulates or regulates, e.g., inhibits, T-cellfunction is PD-1 are provided below.

Provided in Table 7 below are the names of PDCD1 (PD1) RNAi agents(derived from their position in the mouse PDCD1 gene sequence NM008798.2), along with the SEQ ID NOs: 159-206 representing the DNAsequence. Both sense (S) and antisense (AS) sequences are presented as19mer and 21mer sequences are in this table. Also note that the position(PoS, e.g., 176) is derived from the position number in the mouse PDCD1gene sequence NM_008798.2. SEQ ID NOs are indicated in groups of 12 thatcorrespond with “sense 19” SEQ ID NOs: 159-170; “sense 21” SEQ ID NOs:171-182; “asense 21” SEQ ID NOs: 183-194; “asense 19” SEQ ID NOs:195-206.

TABLE 7 Mouse PDCD 1 (PD1) shRNA sequences Position on Target NM_008798.2 region Sense19 Sense21 Asense21 Asense19 176 CDS GGAGGTCCCTCTGGAGGTCC TAGAAGGTGA TAGAAGGTGA CACCTTCTA CTCACCTTCT GGGACCTCCAGGGACCTCC (SEQ ID NO: A G (SEQ ID NO: 159) (SEQ ID NO: (SEQ ID NO: 195)171) 183) 260 CDS CGGAGGATCT GTCGGAGGAT TTCAGCATAA TTCAGCATAA TATGCTGAACTTATGCTGA GATCCTCCGA GATCCTCCG (SEQ ID NO: A C (SEQ ID NO: 160)(SEQ ID NO: (SEQ ID NO: 196) 172) 184) 359 CDS CCCGCTTCCA TGCCCGCTTCTGTATGATCT TGTATGATCT GATCATACA CAGATCATAC GGAAGCGGGC GGAAGCGGG(SEQ ID NO: A A (SEQ ID NO: 161) (SEQ ID NO: (SEQ ID NO: 197) 173) 185)528 CDS GGAGACCTCA CTGGAGACCT ATATCTTGTT ATATCTTGTT ACAAGATAT CAACAAGATAGAGGTCTCCA GAGGTCTCC (SEQ ID NO: T G (SEQ ID NO: 162) (SEQ ID NO:(SEQ ID NO: 198) 174) 186) 581 CDS AAGGCATGGT TCAAGGCATG ATACCAATGAATACCAATGA CATTGGTAT GTCATTGGTA CCATGCCTTG CCATGCCTT (SEQ ID NO: T A(SEQ ID NO: 163) (SEQ ID NO: (SEQ ID NO: 199) 175) 187) 584 CDSGCATGGTCAT AGGCATGGTC ATGATACCAA ATGATACCAA TGGTATCAT ATTGGTATCATGACCATGCC TGACCATGC (SEQ ID NO: T T (SEQ ID NO: 164) (SEQ ID NO:(SEQ ID NO: 200) 176) 188) 588 CDS GGTCATTGGT ATGGTCATTG ATGGTCATTGATGGTCATTG ATCATGAGT GTATCATGAG GTATCATGAG GTATCATGA (SEQ ID NO: T T(SEQ ID NO: 165) (SEQ ID NO: (SEQ ID NO: 201) 177) 189) 609 CDSCCTAGTGGGT GCCCTAGTGG GCCCTAGTGG GCCCTAGTGG ATCCCTGTA GTATCCCTGTGTATCCCTGT GTATCCCTG (SEQ ID NO: A A (SEQ ID NO: 166) (SEQ ID NO:(SEQ ID NO: 202) 178) 190) 919 CDS GAGGATGGAC ATGAGGATGG ATGAGGATGGATGAGGATGG ATTGTTCTT ACATTGTTCTT ACATTGTTCTT ACATTGTTC (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 167) 179) 191) 203) 1021 3′UTRGCATGCAGGC GAGCATGCAG GAGCATGCAG GAGCATGCAG TACAGTTCA GCTACAGTTCGCTACAGTTC GCTACAGTT (SEQ ID NO: A A (SEQ ID NO: 168) (SEQ ID NO:(SEQ ID NO: 204) 180) 192) 1097 3′UTR CCAGCACATG TTCCAGCACA TTCCAGCACATTCCAGCACA CACTGTTGA TGCACTGTTG TGCACTGTTG TGCACTGTT (SEQ ID NO: A A(SEQ ID NO: 169) (SEQ ID NO: (SEQ ID NO: 205) 181) 193) 1101 3′UTRCACATGCACT AGCACATGCA AGCACATGCA AGCACATGCA GTTGAGTGA CTGTTGAGTGCTGTTGAGTG CTGTTGAGT (SEQ ID NO: A A (SEQ ID NO: 170) (SEQ ID NO:(SEQ ID NO: 206) 182) 194)

Provided in Table 8 below are the names of PDCD1 (PD1) RNAi agents(derived from their position in the human PDCD1 gene sequence, alongwith the SEQ ID NOs. 207-254 representing the DNA sequence. Both sense(S) and antisense (AS) sequences are presented as 19mer and 21mersequences. SEQ ID NOs are indicated in groups of 12 that correspond with“sense 19” SEQ ID NOs: 207-218; “sense 21” SEQ ID NOs: 219-230; “asense21” SEQ ID NOs: 231-242; “asense 19” SEQ ID NOs: 243-254.

TABLE 8 Human PDCD1 (PD1) shRNA sequences Position on Target NM_005018.2region Sense19 Asense19 Sense21 Asense21 145 CDS GGCCAGGATG TCTAAGAACCGCGGCCAGGA TCTAAGAACC GTTCTTAGA ATCCTGGCC TGGTTCTTAG ATCCTGGCCG(SEQ ID NO: (SEQ ID NO: A C 207) 219) (SEQ ID NO: (SEQ ID NO: 231) 243)271 CDS GCTTCGTGCT TACCAGTTTA GAGCTTCGTG TACCAGTTTA AAACTGGTA GCACGAAGCCTAAACTGGT GCACGAAGCT (SEQ ID NO: (SEQ ID NO: A C 208) 220) (SEQ ID NO:(SEQ ID NO: 232) 244) 393 CDS GGGCGTGACT TCATGTGGAA ACGGGCGTGATCATGTGGAA TCCACATGA GTCACGCCC CTTCCACATG GTCACGCCCG (SEQ ID NO:(SEQ ID NO: A T 209) 221) (SEQ ID NO: (SEQ ID NO: 233) 245) 1497 3′UTRCAGGCCTAGA TGAAACTTCT TGCAGGCCTA TGAAACTTCT GAAGTTTCA CTAGGCCTGGAGAAGTTTC CTAGGCCTGC (SEQ ID NO: (SEQ ID NO: A A 210) 222) (SEQ ID NO:(SEQ ID NO: 234) 246) 1863 3′UTR CTTGGAACCC TTCAGGAATG TCCTTGGAACTTCAGGAATG ATTCCTGAA GGTTCCAAG CCATTCCTGA GGTTCCAAGG (SEQ ID NO:(SEQ ID NO: A A 211) 223) (SEQ ID NO: (SEQ ID NO: 235) 247) 1866 3′UTRGGAACCCATT AATTTCAGGA TTGGAACCCA AATTTCAGGA CCTGAAATT ATGGGTTCCTTCCTGAAAT ATGGGTTCCA (SEQ ID NO: (SEQ ID NO: T A 212) 224) (SEQ ID NO:(SEQ ID NO: 236) 248) 1867 3′UTR GAACCCATTC TAATTTCAGG TGGAACCCATTAATTTCAGG CTGAAATTA AATGGGTTC TCCTGAAATT AATGGGTTCC (SEQ ID NO:(SEQ ID NO: A A 213) 225) (SEQ ID NO: (SEQ ID NO: 237) 249) 1868 3′UTRAACCCATTCC ATAATTTCAG GGAACCCATT ATAATTTCAG TGAAATTAT GAATGGGTTCCTGAAATTA GAATGGGTTC (SEQ ID NO: (SEQ ID NO: T C 214) 226) (SEQ ID NO:(SEQ ID 238) NO: 250) 1869 3′UTR ACCCATTCCT AATAATTTCA GAACCCATTCAATAATTTCA GAAATTATT GGAATGGGT CTGAAATTAT GGAATGGGTT (SEQ ID NO:(SEQ ID NO: T C 215) 227) (SEQ ID NO: (SEQ ID NO: 239) 251) 1870 3′UTRCCCATTCCTG AAATAATTTC AACCCATTCC AAATAATTTC AAATTATTT AGGAATGGGTGAAATTATT AGGAATGGGT (SEQ ID NO: (SEQ ID NO: T T 216) 228) (SEQ ID NO:(SEQ ID NO: 240) 252) 2079 3′UTR CTGTGGTTCT TAATATAATA CCCTGTGGTTTAATATAATA ATTATATTA GAACCACAG CTATTATATT GAACCACAGG (SEQ ID NO:(SEQ ID NO: A G 217) 229) (SEQ ID NO: (SEQ ID NO: 241) 253) 2109 3′UTRAAATATGAGA TTAGCATGCT TTAAATATGA TTAGCATGCT GCATGCTAA CTCATATTTGAGCATGCTA CTCATATTTA (SEQ ID NO: (SEQ ID NO: A A 218) 230) (SEQ ID NO:(SEQ ID NO: 242) 254)

In one embodiment, the inhibitor of an inhibitory signal can be, e.g.,an antibody or antibody fragment that binds to an inhibitory molecule.For example, the agent can be an antibody or antibody fragment thatbinds to PD1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred toas MDX-010 and MDX-101, and marketed as Yervoy®; Bristol-Myers Squibb;Tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerlyknown as ticilimumab, CP-675,206).). In an embodiment, the agent is anantibody or antibody fragment that binds to TIM3. In an embodiment, theagent is an antibody or antibody fragment that binds to LAG3. Inembodiments, the agent that enhances the activity of a CAR-expressingcell, e.g., inhibitor of an inhibitory molecule, is administered incombination with an allogeneic CAR, e.g., an allogeneic CAR describedherein (e.g., described in the Allogeneic CAR section herein).

PD1 is an inhibitory member of the CD28 family of receptors that alsoincludes CD28, CTLA-4, ICOS, and BTLA. PD1 is expressed on activated Bcells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol8:765-75). Two ligands for PD1, PD-L1 and PD-L2 have been shown todownregulate T cell activation upon binding to PD1 (Freeman et a. 2000 JExp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter etal. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers(Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol.Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094).Immune suppression can be reversed by inhibiting the local interactionof PD1 with PD-L1. Antibodies, antibody fragments, and other inhibitorsof PD1, PD-L1 and PD-L2 are available in the art and may be usedcombination with a CD33 CAR described herein. For example, nivolumab(also referred to as BMS-936558 or MDX1106; Bristol-Myers Squibb) is afully human IgG4 monoclonal antibody which specifically blocks PD1.Nivolumab (clone 5C4) and other human monoclonal antibodies thatspecifically bind to PD1 are disclosed in U.S. Pat. No. 8,008,449 andWO2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1kmonoclonal antibody that binds to PD1Pidilizumab and other humanizedanti-PD1 monoclonal antibodies are disclosed in WO2009/101611.Lambrolizumab (also referred to as MK03475; Merck) is a humanized IgG4monoclonal antibody that binds to PD1. Lambrolizumab and other humanizedanti-PD1 antibodies are disclosed in U.S. Pat. No. 8,354,509 andWO2009/114335. MDPL3280A (Genentech/Roche) is a human Fc optimized IgG1monoclonal antibody that binds to PD-L1. MDPL3280A and other humanmonoclonal antibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743and U.S Publication No.: 20120039906. Other anti-PD-L1 binding agentsinclude YW243.55.570 (heavy and light chain variable regions are shownin SEQ ID NOs 20 and 21 in WO2010/077634) and MDX-1 105 (also referredto as BMS-936559, and, e.g., anti-PD-L1 binding agents disclosed inWO2007/005874). AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed inWO2010/027827 and WO2011/066342), is a PD-L2 Fc fusion soluble receptorthat blocks the interaction between PD1 and B7-H1. Other anti-PD1antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD1antibodies disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and/orUS 20120114649.

TIM3 (T cell immunoglobulin-3) also negatively regulates T cellfunction, particularly in IFN-g-secreting CD4+ T helper 1 and CD8+ Tcytotoxic 1 cells, and plays a critical role in T cell exhaustion.Inhibition of the interaction between TIM3 and its ligands, e.g.,galectin-9 (Gal9), phosphotidylserine (PS), and HMGB1, can increaseimmune response. Antibodies, antibody fragments, and other inhibitors ofTIM3 and its ligands are available in the art and may be usedcombination with a CD19 CAR described herein. For example, antibodies,antibody fragments, small molecules, or peptide inhibitors that targetTIM3 binds to the IgV domain of TIM3 to inhibit interaction with itsligands. Antibodies and peptides that inhibit TIM3 are disclosed inWO2013/006490 and US20100247521. Other anti-TIM3 antibodies includehumanized versions of RMT3-23 (disclosed in Ngiow et al., 2011, CancerRes, 71:3540-3551), and clone 8B.2C12 (disclosed in Monney et al., 2002,Nature, 415:536-541). Bi-specific antibodies that inhibit TIM3 and PD-1are disclosed in US20130156774.

In other embodiments, the agent which enhances the activity of aCAR-expressing cell is a CEACAM inhibitor (e.g., CEACAM-1, CEACAM-3,and/or CEACAM-5 inhibitor). In one embodiment, the inhibitor of CEACAMis an anti-CEACAM antibody molecule. Exemplary anti-CEACAM-1 antibodiesare described in WO 2010/125571, WO 2013/082366 WO 2014/059251 and WO2014/022332, e.g., a monoclonal antibody 34B1, 26H7, and 5F4; or arecombinant form thereof, as described in, e.g., US 2004/0047858, U.S.Pat. No. 7,132,255 and WO 99/052552. In other embodiments, theanti-CEACAM antibody binds to CEACAM-5 as described in, e.g., Zheng etal. PLoS One. 2010 Sep. 2;5(9). pii: e12529(DOI:10:1371/journal.pone.0021146), or crossreacts with CEACAM-1 andCEACAM-5 as described in, e.g., WO 2013/054331 and US 2014/0271618.

Without wishing to be bound by theory, carcinoembryonic antigen celladhesion molecules (CEACAM), such as CEACAM-1 and CEACAM-5, are believedto mediate, at least in part, inhibition of an anti-tumor immuneresponse (see e.g., Markel et al. J Immunol. 2002 Mar. 15;168(6):2803-10; Markel et al. J Immunol. 2006 Nov. 1; 177(9):6062-71;Markel et al. Immunology. 2009 February; 126(2):186-200; Markel et al.Cancer Immunol Immunother. 2010 February; 59(2):215-30; Ortenberg et al.Mol Cancer Ther. 2012 June; 11(6):1300-10; Stern et al. J Immunol. 2005Jun. 1; 174(11):6692-701; Zheng et al. PLoS One. 2010 Sep. 2; 5(9). pii:e12529). For example, CEACAM-1 has been described as a heterophilicligand for TIM-3 and as playing a role in TIM-3-mediated T celltolerance and exhaustion (see e.g., WO 2014/022332; Huang, et al. (2014)Nature doi:10.1038/nature13848). In embodiments, co-blockade of CEACAM-1and TIM-3 has been shown to enhance an anti-tumor immune response inxenograft colorectal cancer models (see e.g., WO 2014/022332; Huang, etal. (2014), supra). In other embodiments, co-blockade of CEACAM-1 andPD-1 reduce T cell tolerance as described, e.g., in WO 2014/059251.Thus, CEACAM inhibitors can be used with the other immunomodulatorsdescribed herein (e.g., anti-PD-1 and/or anti-TIM-3 inhibitors) toenhance an immune response against a cancer, e.g., a melanoma, a lungcancer (e.g., NSCLC), a bladder cancer, a colon cancer an ovariancancer, and other cancers as described herein.

LAG3 (lymphocyte activation gene-3 or CD223) is a cell surface moleculeexpressed on activated T cells and B cells that has been shown to play arole in CD8+ T cell exhaustion. Antibodies, antibody fragments, andother inhibitors of LAG3 and its ligands are available in the art andmay be used combination with a CD19 CAR described herein. For example,BMS-986016 (Bristol-Myers Squib) is a monoclonal antibody that targetsLAG3. IMP701 (Immutep) is an antagonist LAG3 antibody and IMP731(Immutep and GlaxoSmithKline) is a depleting LAG3 antibody. Other LAG3inhibitors include IMP321 (Immutep), which is a recombinant fusionprotein of a soluble portion of LAG3 and Ig that hinds to MHC class IImolecules and activates antigen presenting cells (APC). Other antibodiesare disclosed, e.g., in WO2010/019570.

In some embodiments, the agent which enhances the activity of aCAR-expressing cell can be, e.g., a fusion protein comprising a firstdomain and a second domain, wherein the first domain is an inhibitorymolecule, or fragment thereof, and the second domain is a polypeptidethat is associated with a positive signal, e.g., a polypeptidecomprising an antracellular signaling domain as described herein. Insome embodiments, the polypeptide that is associated with a positivesignal can include a costimulatory domain of CD28, CD27, ICOS, e.g., anintracellular signaling domain of CD28, CD27 and/or ICOS, and/or aprimary signaling domain, e.g., of CD3 zeta, e.g., described herein. Inone embodiment, the fusion protein is expressed by the same cell thatexpressed the CAR. In another embodiment, the fusion protein isexpressed by a cell, e.g., a T cell that does not express a CD33 CAR.

In one embodiment, the agent which enhances activity of a CAR-expressingcell described herein is miR-17-92.

In one embodiment, the agent which enhances activity of a CAR-describedherein is a cytokine. Cytokines have important functions related to Tcell expansion, differentiation, survival, and homeostatis. Cytokinesthat can be administered to the subject receiving a CAR-expressing celldescribed herein include: IL-2, IL-4, IL-7, IL-9, IL-15, IL-18, andIL-21, or a combination thereof. In preferred embodiments, the cytokineadministered is IL-7, IL-15, or IL-21, or a combination thereof. Thecytokine can be administered once a day or more than once a day, e.g.,twice a day, three times a day, or four times a day. The cytokine can beadministered for more than one day, e.g. the cytokine is administeredfor 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or4 weeks. For example, the cytokine is administered once a day for 7days.

In embodiments, the cytokine is administered in combination withCAR-expressing T cells. The cytokine can be administered simultaneouslyor concurrently with the CAR-expressing T cells, e.g., administered onthe same day. The cytokine may be prepared in the same pharmaceuticalcomposition as the CAR-expressing T cells, or may be prepared in aseparate pharmaceutical composition. Alternatively, the cytokine can beadministered shortly after administration of the CAR-expressing T cells,e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days afteradministration of the CAR-expressing T cells. In embodiments where thecytokine is administered in a dosing regimen that occurs over more thanone day, the first day of the cytokine dosing regimen can be on the sameday as administration with the CAR-expressing T cells, or the first dayof the cytokine dosing regimen can be 1 day, 2 days, 3 days, 4 days, 5days, 6 days, or 7 days after administration of the CAR-expressing Tcells. In one embodiment, on the first day, the CAR-expressing T cellsare administered to the subject, and on the second day, a cytokine isadministered once a day for the next 7 days. In a preferred embodiment,the cytokine to be administered in combination with CAR-expressing Tcells is IL-7, IL-15, or IL-21.

In other embodiments, the cytokine is administered a period of timeafter administration of CAR-expressing cells, e.g., at least 2 weeks, 3weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or1 year or more after administration of CAR-expressing cells. In oneembodiment, the cytokine is administered after assessment of thesubject's response to the CAR-expressing cells. For example, the subjectis administered CAR-expressing cells according to the dosage andregimens described herein. The response of the subject to CAR-expressingcell therapy is assessed at 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks,10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, or 1 year or more after administration ofCAR-expressing cells, using any of the methods described herein,including inhibition of tumor growth, reduction of circulating tumorcells, or tumor regression. Subjects that do not exhibit a sufficientresponse to CAR-expressing cell therapy can be administered a cytokine.Administration of the cytokine to the subject that has sub-optimalresponse to the CAR-expressing cell therapy improves CAR-expressing cellefficacy or anti-cancer activity. In a preferred embodiment, thecytokine administered after administration of CAR-expressing cells isIL-7.

COMBINATION WITH a Low, IMMUNE ENHANCING, DOSE OF AN MTOR INHIBITOR

Methods described herein use low, immune enhancing, doses of mTORinhibitors, e.g., allosteric mTOR inhibitors, including rapalogs such asRAD001. Administration of a low, immune enhancing, dose of an mTORinhibitor (e.g., a dose that is insufficient to completely suppress theimmune system, but sufficient to improve immune function) can optimizethe performance of immune effector cells, e.g., T cells orCAR-expressing cells, in the subject. Methods for measuring mTORinhibition, dosages, treatment regimens, and suitable pharmaceuticalcompositions are described in U.S. Patent Application No. 2015/01240036,hereby incorporated by reference.

In an embodiment, administration of a low, immune enhancing, dose of anmTOR inhibitor can result in one or more of the following:

-   -   i) a decrease in the number of PD-1 positive immune effector        cells;    -   ii) an increase in the number of PD-1 negative immune effector        cells;    -   iii) an increase in the ratio of PD-1 negative immune effector        cells/PD-1 positive immune effector cells;    -   iv) an increase in the number of naive T cells;    -   v) an increase in the expression of one or more of the following        markers: CD62L^(high), CD127^(high), CD27⁺, and BCL2, e.g., on        memory T cells, e.g., memory T cell precursors;    -   vi) a decrease in the expression of KLRG1, e.g., on memory T        cells, e.g., memory T cell precursors; or    -   vii) an increase in the number of memory T cell precursors,        e.g., cells with any one or combination of the following        characteristics: increased CD62L^(high), increased CD127^(high),        increased CD27⁺, decreased KLRG1, and increased BCL2;    -   and wherein any of the foregoing, e.g., i), ii), iii), iv), v),        vi), or vii), occurs e.g., at least transiently, e.g., as        compared to a non-treated subject.

In another embodiment, administration of a low, immune enhancing, doseof an mTOR inhibitor results in increased or prolonged proliferation orpersistence of CAR-expressing cells, e.g., in culture or in a subject,e.g., as compared to non-treated CAR-expressing cells or a non-treatedsubject. In embodiments, increased proliferation or persistence isassociated with in an increase in the number of CAR-expressing cells.Methods for measuring increased or prolonged proliferation are describedin Examples 8 and 9. In another embodiment, administration of a low,immune enhancing, dose of an mTOR inhibitor results in increased killingof cancer cells by CAR-expressing cells, e.g., in culture or in asubject, e.g., as compared to non-treated CAR-expressing cells or anon-treated subject. In embodiments, increased killing of cancer cellsis associated with in a decrease in tumor volume. Methods for measuringincreased killing of cancer cells are described in Example 6.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with a low,immune enhancing dose of an mTOR inhibitor, e.g., an allosteric mTORinhibitor, e.g., RAD001, or a catalytic mTOR inhibitor. For example,administration of the low, immune enhancing, dose of the mTOR inhibitorcan be initiated prior to administration of a CAR-expressing celldescribed herein; completed prior to administration of a CAR-expressingcell described herein; initiated at the same time as administration of aCAR-expressing cell described herein; overlapping with administration ofa CAR-expressing cell described herein; or continuing afteradministration of a CAR-expressing cell described herein.

Alternatively or in addition, administration of a low, immune enhancing,dose of an mTOR inhibitor can optimize immune effector cells to beengineered to express a CAR molecule described herein. In suchembodiments, administration of a low, immune enhancing, dose of an mTORinhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or a catalyticinhibitor, is initiated or completed prior to harvest of immune effectorcells, e.g., T cells or NK cells, to be engineered to express a CARmolecule described herein, from a subject.

In another embodiment, immune effector cells, e.g., T cells or NK cells,to be engineered to express a CAR molecule described herein, e.g., afterharvest from a subject, or CAR-expressing immune effector cells, e.g., Tcells or NK cells, e.g., prior to administration to a subject, can becultured in the presence of a low, immune enhancing, dose of an mTORinhibitor.

In an embodiment, administering to the subject a low, immune enhancing,dose of an mTOR inhibitor comprises administering, e.g., once per week,e.g., in an immediate release dosage form, 0.1 to 20, 0.5 to 10, 2.5 to7.5, 3 to 6, or about 5, mgs of RAD001, or a bioequivalent dose thereof.In an embodiment, administering to the subject a low, immune enhancing,dose of an mTOR inhibitor comprises administering, e.g., once per week,e.g., in a sustained release dosage form, 0.3 to 60, 1.5 to 30, 7.5 to22.5, 9 to 18, or about 15 mgs of RAD001, or a bioequivalent dosethereof.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 90%, at least10 but no more than 90%, at least 15, but no more than 90%, at least 20but no more than 90%, at least 30 but no more than 90%, at least 40 butno more than 90%, at least 50 but no more than 90%, at least 60 but nomore than 90%, at least 70 but no more than 90%, at least 5 but no morethan 80%, at least 10 but no more than 80%, at least 15, but no morethan 80%, at least 20 but no more than 80%, at least 30 but no more than80%, at least 40 but no more than 80%, at least 50 but no more than 80%,at least 60 but no more than 80%, at least 5 but no more than 70%, atleast 10 but no more than 70%, at least 15, but no more than 70%, atleast 20 but no more than 70%, at least 30 but no more than 70%, atleast 40 but no more than 70%, at least 50 but no more than 70%, atleast 5 but no more than 60%, at least 10 but no more than 60%, at least15, but no more than 60%, at least 20 but no more than 60%, at least 30but no more than 60%, at least 40 but no more than 60%, at least 5 butno more than 50%, at least 10 but no more than 50%, at least 15, but nomore than 50%, at least 20 but no more than 50%, at least 30 but no morethan 50%, at least 40 but no more than 50%, at least 5 but no more than40%, at least 10 but no more than 40%, at least 15, but no more than40%, at least 20 but no more than 40%, at least 30 but no more than 40%,at least 35 but no more than 40%, at least 5 but no more than 30%, atleast 10 but no more than 30%, at least 15, but no more than 30%, atleast 20 but no more than 30%, or at least 25 but no more than 30%.

The extent of mTOR inhibition can be conveyed as, or corresponds to, theextent of P70 S6 kinase inhibition, e.g., the extent of mTOR inhibitioncan be determined by the level of decrease in P70 S6 kinase activity,e.g., by the decrease in phosphorylation of a P70 S6 kinase substrate.The level of mTOR inhibition can be evaluated by various methods, suchas measuring P70 S6 kinase activity by the Boulay assay, as described inU.S. Patent Application No. 2015/01240036, hereby incorporated byreference, or as described in U.S. Pat. No. 7,727,950, herebyincorporated by reference; measuring the level of phosphorylated S6 bywestern blot; or evaluating a change in the ratio of PD1 negative immuneeffector cells to PD1 positive immune effector cells.

As used herein, the term “mTOR inhibitor” refers to a compound orligand, or a pharmaceutically acceptable salt thereof, which inhibitsthe mTOR kinase in a cell. In an embodiment, an mTOR inhibitor is anallosteric inhibitor. Allosteric mTOR inhibitors include the neutraltricyclic compound rapamycin (sirolimus), rapamycin-related compounds,that is compounds having structural and functional similarity torapamycin including, e.g., rapamycin derivatives, rapamycin analogs(also referred to as rapalogs) and other macrolide compounds thatinhibit mTOR activity. In an embodiment, an mTOR inhibitor is acatalytic inhibitor.

Rapamycin is a known macrolide antibiotic produced by Streptomyceshygroscopicus having the structure shown in Formula A.

See, e.g., McAlpine, J. B., et al., J. Antibiotics (1991) 44: 688;Schreiber, S. L., et al., J. Am. Chem. Soc. (1991) 113: 7433; U.S. Pat.No. 3,929,992. There are various numbering schemes proposed forrapamycin. To avoid confusion, when specific rapamycin analogs are namedherein, the names are given with reference to rapamycin using thenumbering scheme of formula A.

Rapamycin analogs useful in the invention are, for example,0-substituted analogs in which the hydroxyl group on the cyclohexyl ringof rapamycin is replaced by OR₁ in which R₁ is hydroxyalkyl,hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl; e.g. RAD001, alsoknown as everolimus, as described in U.S. Pat. No. 5,665,772 andWO94/09010, the contents of each are incorporated by reference.

Other suitable rapamycin analogs include those substituted at the 26- or28-position. The rapamycin analog may be an epimer of an analogmentioned above, particularly an epimer of an analog substituted inposition 40, 28 or 26, and may optionally be further hydrogenated, e.g.as described in U.S. Pat. No. 6,015,815, WO95/14023 and WO99/15530 thecontents of which are incorporated by reference, e.g. ABT578 also knownas zotarolimus or a rapamycin analog described in U.S. Pat. No.7,091,213, WO98/02441 and WO01/14387 the contents of which areincorporated by reference, e.g. AP23573 also known as ridaforolimus.

Examples of rapamycin analogs suitable for use in the present inventionfrom U.S. Pat. No. 5,665,772 include, but are not limited to,40-O-benzyl-rapamycin, 40-O-(4′-hydroxymethyl)benzyl-rapamycin,40-O-[4′-(1,2-dihydroxyethyl)]benzyl-rapamycin, 40-O-allyl-rapamycin,40-O-[3′-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2′-en-1′-yl]-rapamycin,(2′E,4'S)-40-O-(4′,5′-dihydroxypent-2′-en-1′-yl)-rapamycin,40-O-(2-hydroxy)ethoxycarbonylmethyl-rapamycin,40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin,40-O-(6-hydroxy)hexyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-[(3S)-2,2-dimethyldioxolan-3-yl]methyl-rapamycin,40-O-[(2S)-2,3-dihydroxyprop-1-yl]-rapamycin,40-O-(2-acetoxy)ethyl-rapamycin, 40-O-[2-nicotinoyloxy)ethyl-rapamycin,40-O-[2-(N-morpholino)acetoxy]ethyl-rapamycin,40-042-N-imidazolylacetoxy)ethyl-rapamycin,40-O-[24N-methyl-N′-piperazinyl)acetoxy]ethyl-rapamycin,39-O-desmethyl-39,40-O,O-ethylene-rapamycin,(26R)-26-dihydro-40-O-(2-hydroxy)ethyl-rapamycin,40-O-(2-aminoethyl)-rapamycin, 40-O-(2-acetaminoethyl)-rapamycin,40-O-(2-nicotinamidoethyl)-rapamycin,40-O-[(2-(N-methyl-imidazo-2′-ylcarbethoxamido)ethyl)-rapamycin,40-O-(2-ethoxycarbonylaminoethyl)-rapamycin,40-O-(2-tolylsulfonamidoethyl)-rapamycin and40-O-[2-(4′,5′-dicarboethoxy-1′,2′,3′-triazol-1′-yl)-ethyl]-rapamycin.

Other rapamycin analogs useful in the present invention are analogswhere the hydroxyl group on the cyclohexyl ring of rapamycin and/or thehydroxy group at the 28 position is replaced with an hydroxyester groupare known, for example, rapamycin analogs found in US RE44,768, e.g.temsirolimus.

Other rapamycin analogs useful in the preset invention include thosewherein the methoxy group at the 16 position is replaced with anothersubstituent, preferably (optionally hydroxy-substituted) alkynyloxy,benzyl, orthomethoxybenzyl or chlorobenzyl and/or wherein the mexthoxygroup at the 39 position is deleted together with the 39 carbon so thatthe cyclohexyl ring of rapamycin becomes a cyclopentyl ring lacking the39 position methyoxy group; e.g. as described in WO95/16691 andWO96/41807, the contents of which are incorporated by reference. Theanalogs can be further modified such that the hydroxy at the 40-positionof rapamycin is alkylated and/or the 32-carbonyl is reduced.

Rapamycin analogs from WO95/16691 include, but are not limited to,16-demthoxy-16-(pent-2-ynyl)oxy-rapamycin,16-demthoxy-16-(but-2-ynyl)oxy-rapamycin,16-demthoxy-16-(propargyl)oxy-rapamycin,16-demethoxy-16-(4-hydroxy-but-2-ynyl)oxy-rapamycin,16-demthoxy-16-benzyloxy-40-O-(2-hydroxyethyl)-rapamycin,16-demthoxy-16-benzyloxy-rapamycin,16-demethoxy-16-ortho-methoxybenzyl-rapamycin,16-demethoxy-40-O-(2-methoxyethyl)-16-pent-2-ynyl)oxy-rapamycin,39-demethoxy-40-desoxy-39-formyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-hydroxymethyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-carboxy-42-nor-rapamycin,39-demethoxy-40-desoxy-39-(4-methyl-piperazin-1-yl)carbonyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-(morpholin-4-yl)carbonyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-[N-methyl,N-(2-pyridin-2-yl-ethyl)]carbamoyl-42-nor-rapamycin and39-demethoxy-40-desoxy-394p-toluenesulfonylhydrazonomethyl)-42-nor-rapamycin.

Rapamycin analogs from WO96/41807 include, but are not limited to,32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-rapamycin,16-O-pent-2-ynyl-32-deoxo-40-O-(2-hydroxy-ethyl)-rapamycin,16-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,32(S)-dihydro-40-O-(2-methoxy)ethyl-rapamycin and32(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin.

Another suitable rapamycin analog is umirolimus as described inUS2005/0101624 the contents of which are incorporated by reference.

RAD001, otherwise known as everolimus (Afinitor®), has the chemical name(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentaone,as described in U.S. Pat. No. 5,665,772 and WO94/09010, the contents ofeach are incorporated by reference.

Further examples of allosteric mTOR inhibitors include sirolimus(rapamycin, AY-22989),40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin (alsocalled temsirolimus or CCI-779) and ridaforolimus (AP-23573/MK-8669).Other examples of allosteric mTorr inhibitors include zotarolimus(ABT578) and umirolimus.

Alternatively or additionally, catalytic, ATP-competitive mTORinhibitors have been found to target the mTOR kinase domain directly andtarget both mTORC1 and mTORC2. These are also more effective inhibitorsof mTORC1 than such allosteric mTOR inhibitors as rapamycin, becausethey modulate rapamycin-resistant mTORC1 outputs such as 4EBP1-T37/46phosphorylation and cap-dependent translation.

Catalytic inhibitors include: BEZ235 or2-methyl-244-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrile,or the monotosylate salt form (the synthesis of BEZ235 is described inWO2006/122806); CCG168 (otherwise known as AZD-8055, Chresta, C. M., etal., Cancer Res, 2010, 70(1), 288-298) which has the chemical name{5-[2,4-bis-((S)-3-methyl-morpholin-4-yl)-pyrido[2,3d]pyrimidin-7-yl]-2-methoxy-phenyl-methanol;3-[2,4-bis[(35)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methylbenzamide(WO09104019);3-(2-aminobenzo[d]oxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine(WO10051043 and WO2013023184); AN-(3-(N-(3-((3,5-dimethoxyphenyl)amino)quinoxaline-2-yl)sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide(WO07044729 and WO12006552); PKI-587 (Venkatesan, A. M., J. Med. Chem.,2010, 53, 2636-2645) which has the chemical name1-[4-[4-(dimethylamino)piperidine-1-carbonyl]phenyl]-3-[4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyflurea;GSK-2126458 (ACS Med. Chem. Lett., 2010, 1, 39-43) which has thechemical name2,4-difluoro-N-{2-methoxy-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide;5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine(WO10114484); and(E)-N-(8-(6-amino-5-(trifluoromethyl)pyridin-3-yl)-1-(6-(2-cyanopropan-2-yl)pyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-ylidene)cyanamide (WO12007926).

Further examples of catalytic mTOR inhibitors include8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one (WO2006/122806) and Ku-0063794 (Garcia-Martinez J M, etal., Biochem J., 2009, 421(1), 29-42. Ku-0063794 is a specific inhibitorof the mammalian target of rapamycin (mTOR).) WYE-354 is another exampleof a catalytic mTOR inhibitor (Yu K, et al. (2009). Biochemical,Cellular, and In vivo Activity of Novel ATP-Competitive and SelectiveInhibitors of the Mammalian Target of Rapamycin. Cancer Res. 69(15):6232-6240).

mTOR inhibitors useful according to the present invention also includeprodrugs, derivatives, pharmaceutically acceptable salts, or analogsthereof of any of the foregoing.

mTOR inhibitors, such as RAD001, may be formulated for delivery based onwell-established methods in the art based on the particular dosagesdescribed herein. In particular, U.S. Pat. No. 6,004,973 (incorporatedherein by reference) provides examples of formulations useable with themTOR inhibitors described herein.

Methods and Biomarkers for Evaluating CAR-Effectiveness or SampleSuitability

In another aspect, the invention features a method of evaluating ormonitoring the effectiveness of a CAR-expressing cell therapy (e.g., aCD33CAR therapy), in a subject (e.g., a subject having a cancer, e.g., ahematological cancer), or the suitability of a sample (e.g., anapheresis sample) for a CAR therapy (e.g., a CD33CAR therapy). Themethod includes acquiring a value of effectiveness to the CAR therapy,or sample suitability, wherein said value is indicative of theeffectiveness or suitability of the CAR-expressing cell therapy.

In embodiments, the value of effectiveness to the CAR therapy, or samplesuitability, comprises a measure of one, two, three, four, five, six ormore (all) of the following:

(i) the level or activity of one, two, three, or more (e.g., all) ofresting T_(EFF) cells, resting T_(REG) cells, younger T cells (e.g.,younger CD4 or CD8 cells, or gamma/delta T cells), or early memory Tcells, or a combination thereof, in a sample (e.g., an apheresis sampleor a manufactured CAR-expressing cell product sample);

(ii) the level or activity of one, two, three, or more (e.g., all) ofactivated T_(EFF) cells, activated T_(REG) cells, older T cells (e.g.,older CD4 or CD8 cells), or late memory T cells, or a combinationthereof, in a sample (e.g., an apheresis sample or a manufacturedCAR-expressing cell product sample);

(iii) the level or activity of an immune cell exhaustion marker, e.g.,one, two or more immune checkpoint inhibitors (e.g., PD-1, PD-L1, TIM-3and/or LAG-3) in a sample (e.g., an apheresis sample or a manufacturedCAR-expressing cell product sample). In one embodiment, an immune cellhas an exhausted phenotype, e.g., co-expresses at least two exhaustionmarkers, e.g., co-expresses PD-1 and TIM-3. In other embodiments, animmune cell has an exhausted phenotype, e.g., co-expresses at least twoexhaustion markers, e.g., co-expresses PD-1 and LAG-3;

(iv) the level or activity of CD27 and/or CD45RO− (e.g., CD27+CD45RO−)immune effector cells, e.g., in a CD4+ or a CD8+ T cell population, in asample (e.g., an apheresis sample or a manufactured CAR-expressing cellproduct sample);

(v) the level or activity of one, two, three, four, five, ten, twenty ormore of the biomarkers chosen from CCL20, IL-17a and/or IL-6, PD-1,PD-L1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1;

(vi) a cytokine level or activity (e.g., quality of cytokine reportoire)in a CAR-expressing cell product sample, e.g., CAR33-expressing cellproduct sample; or

(vii) a transduction efficiency of a CAR-expressing cell in amanufactured CAR-expressing cell product sample.

In some embodiments of any of the methods disclosed herein, theCAR-expressing cell therapy comprises a plurality (e.g., a population)of CAR-expressing immune effector cells, e.g., a plurality (e.g., apopulation) of T cells or NK cells, or a combination thereof. In oneembodiment, the CAR-expressing cell therapy is a CD33CAR therapy.

In some embodiments of any of the methods disclosed herein, the measureof one or more of (i)-(vii) is obtained from an apheresis sampleacquired from the subject. The apheresis sample can be evaluated priorto infusion or re-infusion.

In some embodiments of any of the methods disclosed herein, the measureof one or more of (i)-(vii) is obtained from a manufacturedCAR-expressing cell product sample, e.g., CD33CAR-expressing cellproduct sample. The manufactured CAR-expressing cell product can beevaluated prior to infusion or re-infusion.

In some embodiments of any of the methods disclosed herein, the subjectis evaluated prior to receiving, during, or after receiving, theCAR-expressing cell therapy.

In some embodiments of any of the methods disclosed herein, the measureof one or more of (i)-(vii) evaluates a profile for one or more of geneexpression, flow cytometry or protein expression.

In some embodiments of any of the methods disclosed herein, the methodfurther comprises identifying the subject as a responder, anon-responder, a relapser or a non-relapser, based on a measure of oneor more of (i)-(vii).

In some embodiments of any of the methods disclosed herein, a responder(e.g., a complete responder) has, or is identified as having, a greaterlevel or activity of one, two, or more (all) of GZMK, PPF1BP2, or naïveT cells as compared to a non-responder.

In some embodiments of any of the methods disclosed herein, anon-responder has, or is identified as having, a greater level oractivity of one, two, three, four, five, six, seven, or more (e.g., all)of IL22, IL-2RA, IL-21, IRF8, IL8, CCL17, CCL22, effector T cells, orregulatory T cells, as compared to a responder.

In an embodiment, a relapser is a patient having, or who is identifiedas having, an increased level of expression of one or more of (e.g., 2,3, 4, or all of) the following genes, compared to non relapsers:MIR199A1, MIR1203, uc021ovp, ITM2C, and HLA-DQB1 and/or a decreasedlevels of expression of one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or all of) the following genes, compared to non relapsers:PPIAL4D, TTTY10, TXLNG2P, MIR4650-1, KDMSD, USP9Y, PRKY, RPS4Y2, RPS4Y1,NCRNA00185, SULT1E1, and EIF1AY.

In some embodiments of any of the methods disclosed herein, a completeresponder has, or is identified as having, a greater, e.g., astatistically significant greater, percentage of CD8+ T cells comparedto a reference value, e.g., a non-responder percentage of CD8+ T cells.

In some embodiments of any of the methods disclosed herein, a completeresponder has, or is identified as having, a greater percentage of CD27+CD45RO− immune effector cells, e.g., in the CD8+ population, compared toa reference value, e.g., a non-responder number of CD27+CD45RO− immuneeffector cells.

In some embodiments of any of the methods disclosed herein, a completeresponder or a partial responder has, or is identified as having, agreater, e.g., a statistically significant greater, percentage of CD4+ Tcells compared to a reference value, e.g., a non-responder percentage ofCD4+ T cells.

In some embodiments of any of the methods disclosed herein, a completeresponder has, or is identified as having, a greater percentage of one,two, three, or more (e.g., all) of resting T_(EFF) cells, restingT_(REG) cells, younger T cells (e.g., younger CD4 or CD8 cells, orgamma/delta T cells), or early memory T cells, or a combination thereof,compared to a reference value, e.g., a non-responder number of restingT_(EFF) cells, resting T_(REG) cells, younger T cells (e.g., younger CD4or CD8 cells), or early memory T cells.

In some embodiments of any of the methods disclosed herein, anon-responder has, or is identified as having, a greater percentage ofone, two, three, or more (e.g., all) of activated T_(EFF) cells,activated T_(REG) cells, older T cells (e.g., older CD4 or CD8 cells),or late memory T cells, or a combination thereof, compared to areference value, e.g., a responder number of activated T_(EFF) cells,activated T_(REG) cells, older T cells (e.g., older CD4 or CD8 cells),or late memory T cells.

In some embodiments of any of the methods disclosed herein, anon-responder has, or is identified as having, a greater percentage ofan immune cell exhaustion marker, e.g., one, two or more immunecheckpoint inhibitors (e.g., PD-1, PD-L1, TIM-3 and/or LAG-3). In oneembodiment, a non-responder has, or is identified as having, a greaterpercentage of PD-1, PD-L1, or LAG-3 expressing immune effector cells(e.g., CD4+ T cells and/or CD8+ T cells) (e.g., CAR-expressing CD4+cells and/or CD8+ T cells) compared to the percentage of PD-1 or LAG-3expressing immune effector cells from a responder.

In one embodiment, a non-responder has, or is identified as having, agreater percentage of immune cells having an exhausted phenotype, e.g.,immune cells that co-express at least two exhaustion markers, e.g.,co-expresses PD-1, PD-L1 and/or TIM-3. In other embodiments, anon-responder has, or is identified as having, a greater percentage ofimmune cells having an exhausted phenotype, e.g., immune cells thatco-express at least two exhaustion markers, e.g., co-expresses PD-1 andLAG-3.

In some embodiments of any of the methods disclosed herein, anon-responder has, or is identified as having, a greater percentage ofPD-1/PD-L1+/LAG-3+ cells in the CAR-expressing cell population (e.g., aCD33CAR+ cell population) compared to a responder (e.g., a completeresponder) to the CAR-expressing cell therapy.

In some embodiments of any of the methods disclosed herein, a partialresponder has, or is identified as having, a higher percentages ofPD-1/PD-L1+/LAG-3+ cells, than a responder, in the CAR-expressing cellpopulation (e.g., a CD33CAR+ cell population).

In some embodiments of any of the methods disclosed herein, anon-responder has, or is identified as having, an exhausted phenotype ofPD1/PD-L1+ CAR+ and co-expression of LAG3 in the CAR-expressing cellpopulation (e.g., a CD33CAR+ cell population).

In some embodiments of any of the methods disclosed herein, anon-responder has, or is identified as having, a greater percentage ofPD-1/PD-L1+/TIM-3+ cells in the CAR-expressing cell population (e.g., aCD33CAR+ cell population) compared to the responder (e.g., a completeresponder).

In some embodiments of any of the methods disclosed herein, a partialresponders has, or is identified as having, a higher percentage ofPD-1/PD-L1+/TIM-3+ cells, than responders, in the CAR-expressing cellpopulation (e.g., a CD33CAR+ cell population).

In some embodiments of any of the methods disclosed herein, the presenceof CD8+CD27+CD45RO− T cells in an apheresis sample is a positivepredictor of the subject response to a CAR-expressing cell therapy(e.g., a CD33CAR therapy).

In some embodiments of any of the methods disclosed herein, a highpercentage of PD1+ CAR+ and LAG3+or TIM3+ T cells in an apheresis sampleis a poor prognostic predictor of the subject response to aCAR-expressing cell therapy (e.g., a CD33CAR therapy).

In some embodiments of any of the methods disclosed herein, theresponder (e.g., the complete or partial responder) has one, two, threeor more (or all) of the following profile:

(i) has a greater number of CD27+ immune effector cells compared to areference value, e.g., a non-responder number of CD27+ immune effectorcells;

(ii) (i) has a greater number of CD8+ T cells compared to a referencevalue, e.g., a non-responder number of CD8+ T cells;

(iii) has a lower number of immune cells expressing one or morecheckpoint inhibitors, e.g., a checkpoint inhibitor chosen from PD-1,PD-L1, LAG-3, TIM-3, or KLRG-1, or a combination, compared to areference value, e.g., a non-responder number of cells expressing one ormore checkpoint inhibitors; or

(iv) has a greater number of one, two, three, four or more (all) ofresting T_(EFF) cells, resting T_(REG) cells, naïve CD4 cells,unstimulated memory cells or early memory T cells, or a combinationthereof, compared to a reference value, e.g., a non-responder number ofresting T_(EFF) cells, resting T_(REG) cells, naïve CD4 cells,unstimulated memory cells or early memory T cells.

In some embodiments of any of the methods disclosed herein, the cytokinelevel or activity of (vi) is chosen from one, two, three, four, five,six, seven, eight, or more (or all) of cytokine CCL20/MIP3a, IL17A, IL6,GM-CSF, IFNγ, IL10, IL13, IL2, IL21, IL4, IL5, IL9 or TNFα, or acombination thereof. The cytokine can be chosen from one, two, three,four or more (all) of IL-17a, CCL20, IL2, IL6, or TNFα. In oneembodiment, an increased level or activity of a cytokine is chosen fromone or both of IL-17a and CCL20, is indicative of increasedresponsiveness or decreased relapse.

In some embodiments of any of the methods disclosed herein, atransduction efficiency of 15% or higher in (vii) is indicative ofincreased responsiveness or decreased relapse.

In some embodiments of any of the methods disclosed herein, atransduction efficiency of less than 15% in (vii) is indicative ofdecreased responsiveness or increased relapse.

In embodiments, the responder, a non-responder, a relapser or anon-relapser identified by the methods herein can be further evaluatedaccording to clinical criteria. For example, a complete responder has,or is identified as, a subject having a disease, e.g., a cancer, whoexhibits a complete response, e.g., a complete remission, to atreatment. A complete response may be identified, e.g., using the NCCNGuidelines or Cheson et al, J Clin Oncol 17:1244 (1999) and Cheson etal., “Revised Response Criteria for Malignant Lymphoma”, J Clin Oncol25:579-586 (2007) (both of which are incorporated by reference herein intheir entireties), as described herein. A partial responder has, or isidentified as, a subject having a disease, e.g., a cancer, who exhibitsa partial response, e.g., a partial remission, to a treatment. A partialresponse may be identified, e.g., using the NCCN Guidelines®, or Chesoncriteria as described herein. A non-responder has, or is identified as,a subject having a disease, e.g., a cancer, who does not exhibit aresponse to a treatment, e.g., the patient has stable disease orprogressive disease. A non-responder may be identified, e.g., using theNCCN Guidelines®, or Cheson criteria as described herein.

Alternatively, or in combination with the methods disclosed herein,responsive to said value, performing one, two, three four or more of:

administering e.g., to a responder or a non-relapser, a CAR-expressingcell therapy;

administered an altered dosing of a CAR-expressing cell therapy;

altering the schedule or time course of a CAR-expressing cell therapy;

administering, e.g., to a non-responder or a partial responder, anadditional agent in combination with a CAR-expressing cell therapy,e.g., a checkpoint inhibitor, e.g., a checkpoint inhibitor describedherein;

administering to a non-responder or partial responder a therapy thatincreases the number of younger T cells in the subject prior totreatment with a CAR-expressing cell therapy;

modifying a manufacturing process of a CAR-expressing cell therapy,e.g., enriching for younger T cells prior to introducing a nucleic acidencoding a CAR, or increasing the transduction efficiency, e.g., for asubject identified as a non-responder or a partial responder;

administering an alternative therapy, e.g., for a non-responder orpartial responder or relapser; or

if the subject is, or is identified as, a non-responder or a relapser,decreasing the T_(REG) cell population and/or T_(REG) gene signature,e.g., by one or more of CD25 depletion, administration ofcyclophosphamide, anti-GITR antibody, or a combination thereof.

In certain embodiments, the subject is pre-treated with an anti-GITRantibody. In certain embodiment, the subject is treated with ananti-GITR antibody prior to infusion or re-infusion.

Biopolymer Delivery Methods

In some embodiments, one or more CAR-expressing cells as disclosedherein can be administered or delivered to the subject via a biopolymerscaffold, e.g., a biopolymer implant.

Biopolymer scaffolds can support or enhance the delivery, expansion,and/or dispersion of the CAR-expressing cells described herein. Abiopolymer scaffold comprises a biocompatible (e.g., does notsubstantially induce an inflammatory or immune response) and/or abiodegradable polymer that can be naturally occurring or synthetic.

Examples of suitable biopolymers include, but are not limited to, agar,agarose, alginate, alginate/calcium phosphate cement (CPC),beta-galactosidase (β-GAL), (1,2,3,4,6-pentaacetyl a-D-galactose),cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acidcollagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate)(PHBHHx), poly(lactide), poly(caprolactone) (PCL),poly(lactide-co-glycolide) (PLG), polyethylene oxide (PEO),poly(lactic-co-glycolic acid) (PLGA), polypropylene oxide (PPO),polyvinyl alcohol) (PVA), silk, soy protein, and soy protein isolate,alone or in combination with any other polymer composition, in anyconcentration and in any ratio. The biopolymer can be augmented ormodified with adhesion- or migration-promoting molecules, e.g.,collagen-mimetic peptides that bind to the collagen receptor oflymphocytes, and/or stimulatory molecules to enhance the delivery,expansion, or function, e.g., anti-cancer activity, of the cells to bedelivered. The biopolymer scaffold can be an injectable, e.g., a gel ora semi-solid, or a solid composition.

In some embodiments, CAR-expressing cells described herein are seededonto the biopolymer scaffold prior to delivery to the subject. Inembodiments, the biopolymer scaffold further comprises one or moreadditional therapeutic agents described herein (e.g., anotherCAR-expressing cell, an antibody, or a small molecule) or agents thatenhance the activity of a CAR-expressing cell, e.g., incorporated orconjugated to the biopolymers of the scaffold. In embodiments, thebiopolymer scaffold is injected, e.g., intratumorally, or surgicallyimplanted at the tumor or within a proximity of the tumor sufficient tomediate an anti-tumor effect. Additional examples of biopolymercompositions and methods for their delivery are described in Stephan etal., Nature Biotechnology, 2015, 33:97-101; and WO2014/110591.

Pharmaceutical Compositions and Treatments

Pharmaceutical compositions of the present invention may comprise aCAR-expressing cell, e.g., a plurality of CAR-expressing cells, asdescribed herein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are in one aspect formulated forintravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

In one embodiment, the pharmaceutical composition is substantially freeof, e.g., there are no detectable levels of a contaminant, e.g.,selected from the group consisting of endotoxin, mycoplasma, replicationcompetent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residualanti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum,bovine serum albumin, bovine serum, culture media components, vectorpackaging cell or plasmid components, a bacterium and a fungus. In oneembodiment, the bacterium is at least one selected from the groupconsisting of Alcaligenes faecalis, Candida albicans, Escherichia coli,Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa,Staphylococcus aureus, Streptococcus pneumonia, and Streptococcuspyogenes group A.

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, in some instances 10⁵ to 10⁶cells/kg body weight, including all integer values within those ranges.T cell compositions may also be administered multiple times at thesedosages. The cells can be administered by using infusion techniques thatare commonly known in immunotherapy (see, e.g., Rosenberg et al., NewEng. J. of Med. 319:1676, 1988).

In certain aspects, it may be desired to administer activated T cells toa subject and then subsequently redraw blood (or have an apheresisperformed), activate T cells therefrom according to the presentinvention, and reinfuse the patient with these activated and expanded Tcells. This process can be carried out multiple times every few weeks.In certain aspects, T cells can be activated from blood draws of from 10cc to 400 cc. In certain aspects, T cells are activated from blood drawsof 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one aspect, the T cell compositions of the presentinvention are administered to a patient by intradermal or subcutaneousinjection. In one aspect, the CAR-expressing cell (e.g., T cell or NKcell) compositions of the present invention are administered by i.v.injection. The compositions of CAR-expressing cells (e.g., T cells or NKcells) may be injected directly into a tumor, lymph node, or site ofinfection.

In a particular exemplary aspect, subjects may undergo leukapheresis,wherein leukocytes are collected, enriched, or depleted ex vivo toselect and/or isolate the cells of interest, e.g., immune effector cells(e.g., T cells or NK cells). These immune effector cell (e.g., T cell orNK cell) isolates may be expanded by methods known in the art andtreated such that one or more CAR constructs of the invention may beintroduced, thereby creating a CAR-expressing cell (e.g., CAR T cell orCAR-expressing NK cell) of the invention. Subjects in need thereof maysubsequently undergo standard treatment with high dose chemotherapyfollowed by peripheral blood stem cell transplantation. In certainaspects, following or concurrent with the transplant, subjects receivean infusion of the expanded CAR-expressing cells (e.g., T cells or NKcells) of the present invention. In an additional aspect, expanded cellsare administered before or following surgery.

In embodiments, lymphodepletion is performed on a subject, e.g., priorto administering one or more cells that express a CAR described herein,e.g., a CD33-binding CAR described herein. In embodiments, thelymphodepletion comprises administering one or more of melphalan,cytoxan, cyclophosphamide, and fludarabine.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

In one embodiment, the CAR is introduced into immune effector cells(e.g., T cells or NK cells), e.g., using in vitro transcription, and thesubject (e.g., human) receives an initial administration of CAR immuneeffector cells (e.g., T cells, NK cells) of the invention, and one ormore subsequent administrations of the CAR immune effector cells (e.g.,T cells, NK cells) of the invention, wherein the one or more subsequentadministrations are administered less than 15 days, e.g., 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previousadministration. In one embodiment, more than one administration of theCAR immune effector cells (e.g., T cells, NK cells) of the invention areadministered to the subject (e.g., human) per week, e.g., 2, 3, or 4administrations of the CAR immune effector cells (e.g., T cells, NKcells) of the invention are administered per week. In one embodiment,the subject (e.g., human subject) receives more than one administrationof the CAR immune effector cells (e.g., T cells, NK cells) per week(e.g., 2, 3 or 4 administrations per week) (also referred to herein as acycle), followed by a week of no CAR immune effector cells (e.g., Tcells, NK cells) administrations, and then one or more additionaladministration of the CAR immune effector cells (e.g., T cells, NKcells) (e.g., more than one administration of the CAR immune effectorcells (e.g., T cells, NK cells) per week) is administered to thesubject. In another embodiment, the subject (e.g., human subject)receives more than one cycle of CAR immune effector cells (e.g., Tcells, NK cells), and the time between each cycle is less than 10, 9, 8,7, 6, 5, 4, or 3 days. In one embodiment, the CAR immune effector cells(e.g., T cells, NK cells) are administered every other day for 3administrations per week. In one embodiment, the CAR immune effectorcells (e.g., T cells, NK cells) of the invention are administered for atleast two, three, four, five, six, seven, eight or more weeks.

In one aspect, CD33 CAR-expressing cells (e.g., CD33 CARTs or CD33CAR-expressing NK cells) are generated using lentiviral viral vectors,such as lentivirus. CAR-expressing cells (e.g., CARTs or CAR-expressingNK cells) generated that way will have stable CAR expression.

In one aspect, CAR-expressing cells, e.g., CARTs, are generated using aviral vector such as a gammaretroviral vector, e.g., a gammaretroviralvector described herein. CARTs generated using these vectors can havestable CAR expression.

In one aspect, CAR-expressing cells (e.g., CARTs or CAR-expressing NKcells) transiently express CAR vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15 days after transduction. Transient expression of CARs can beeffected by RNA CAR vector delivery. In one aspect, the CAR RNA istransduced into the cell, e.g., T cell or NK cell, by electroporation.

A potential issue that can arise in patients being treated usingtransiently expressing CAR-expressing cells (e.g., CARTs orCAR-expressing NK cells) (particularly with murine scFv bearingCAR-expressing cells (e.g., CARTs or CAR-expressing NK cells)) isanaphylaxis after multiple treatments.

Without being bound by this theory, it is believed that such ananaphylactic response might be caused by a patient developing humoralanti-CAR response, i.e., anti-CAR antibodies having an anti-IgE isotype.It is thought that a patient's antibody producing cells undergo a classswitch from IgG isotype (that does not cause anaphylaxis) to IgE isotypewhen there is a ten to fourteen day break in exposure to antigen.

If a patient is at high risk of generating an anti-CAR antibody responseduring the course of transient CAR therapy (such as those generated byRNA transductions), CAR-expressing cell (e.g., CART or CAR-expressing NKcell) infusion breaks should not last more than ten to fourteen days.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples specifically point out various aspects of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure.

Example 1: Humanized CAR Constructs

CD33 levels were measured in primary patent samples of patients havingAML by flow cytometry using a commercially available antibody (cloneHIM3-4, eBioscience; or clone WM53, Biolegend). The results presentedherein demonstrate CD33 was expressed in many primary patient sampleswith AML (AML blasts were gated using standard side scatter ^(low)CD45^(dim) characteristics); n=35-46 per group).

A schematic representation of CAR constructs used this Example is shownin FIG. 3. All are second generation CARs using 41BB and CD3zetasignaling. The scFv of CART33 was derived from clone MY9-6.

In Vitro Activity of CART33

The experiment described herein measured CART33-mediated T celldegranulation. CART33-transduced and UTD T cells were incubated with theCD33+ cell line MOLM14 and a control ALL cell line NALM6 and CD107adegranulation was measured by flow cytometry. Expression of both murineand humanized CART33 constructs elicited specific degranulation in thepresence of MOLM14 (P<0.001) (FIGS. 4A-4B).

The experiment described herein measured cytokine production in responseto CART33. Both humanized and murine CART33 expressing T cells producedcytokine after incubation with MOLM14 (FIGS. 5A-5B). Intracellular tumornecrosis alpha and interferon gamma were measured by flow cytometry.

The experiment described herein measured proliferation of CART123- andCART33-expressing T cells. Humanized CART33 and murine CART33proliferation was measured in response to MOLM14. Results are shown inFIG. 6. T cells were labeled with CFSE and incubated under controlconditions or with MOLM14 for 120 hours. Un-proliferated T cellsretained a single bright peak of CFSE expression (by green fluorescencein the FITC channel), whereas proliferating CART cells had more than oneCFSE peak and expression that was lower than baseline.

The experiment described herein measured specific killing of CART123,humanized CART33- and murine CART33-expressing T cells. T cells wereincubated with MOLM14 or NALM6 (control) for 24 hours. Expression ofhuCART33 resulted in significantly more specific killing compared tomurine CART33 at low E:T ratios (FIG. 7). Killing was measured using aflow cytometry based assay after CF SE-labeling of the tumor cells (e.g.Cao et al, Cytometry Part A 2010; 7&A:534-545) or by incubating CARTcells with luciferase-expressing target cells at variouseffector-to-target ratios for up to 20 hours, followed by opticalimaging for photons emitted by the target cells. In this latter assay,number of live target cells correlates positively with the number ofphotons emitted.

The cytokine profile of humanized CART33-, murine CART33-,CART123-expressing T cells was determined after incubation for 24 hourswith either T cell media alone, PMA/Ionomycin, MOLM14 or NALM6 (controlcell line) (FIG. 8), using a 30-plex Luminex kit (Invitrogen).

CART33 (IgG4 Hinge) and CART33 (CD8 Hinge) have Equivalent In VitroActivity

The experiment described herein measured degranulation. CART33 (IgG4hinge), CART33 (CD8 hinge), CART123, and untransduced T cells wereincubated with the CD33+ cell line MOLM14 and CD107a degranulation wasmeasured by flow cytometry. The results presented herein demonstratethat both CART33 constructs underwent specific degranulation in thepresence of MOLM14 (FIG. 8).

The experiment described herein measured cytokine production. BothCART33 constructs and CART123 specifically induced cytokine productionafter incubation with the MOLM14 cell line (FIG. 9). Intracellular tumornecrosis alpha, MIP1a and interferon gamma were measured by flowcytometry.

The experiment described herein measured proliferation of controluntransduced, CART33- (IgG4 hinge), CART33- (CD8 hinge), orCART123-expressing T cells in response to MOLM14 (FIG. 10). T cells werelabeled with CFSE and incubated with MOLM14 for 120 hours. Proliferationwas measured by CFSE dilution. Unproliferated T cells stain brightlywith CFSE and show a single peak. One cell division is seen as twopeaks, two cell divisions as three peaks, etc.

Equivalent In Vivo Anti-Tumor Effect of CART33-CD8H, CART33-IgG4H, andCART123

To compare in vivo anti-tumor effect of CART33-CD8H, CART33-IgG4H, andCART123, NOD-SCID-common gamma chain knockout (NSG) mice were injectedwith the AML cell line MOLM14 1×10⁶ i.v. and imaged for engraftmentafter 6 days. On day 7, mice were treated with T cells expressing CART33(IgG4 hinge), CART33 (CD8 hinge), CART123, or control vehicle(untransduced cells). Total number of T cells injected was 2×10⁶ IV. Themice were followed with serial weekly imaging to assess tumor burden(FIG. 11).

Data for tumor burden over time was obtained by bioluminescent imaging(BLI). Data from one experiment (n=5 per group), representative of 4independent experiments of the mice is shown in FIG. 12. The resultspresented herein demonstrate equivalent in vivo anti-tumor effect ofCART33-CD8H, CART33-IgG4H, and CART123 T cells.

CART33 and CART123 Produce Equivalent Eradication of Primary AML In Vivo

To compare CART33 and CART123 eradication of primary AML in vivo, NSGmice transgenic for the human cytokines IL3/GM-CSF/SCF (NSGS mice) wereinjected with a primary AML sample at 5×10⁶ i.v. Engraftment wasconfirmed by retro-orbital bleeding after 2-4 weeks and then mice weretreated with CART33, CART123, or control vehicle (untransduced cells).Total number of T cells injected was 1×10⁵ i.v. The mice were followedwith serial retro-orbital bleedings to assess the burden of AML (FIG.13).

Analysis of peripheral blood from mice treated with UTD, CART33 orCART123 was performed at baseline, day 14 and day +70 (FIGS. 14A-14C).Blood was obtained from the retro-orbital sinus of anesthetized miceusing standard techniques. A standard volume of 50-60 ul blood was thenlysed in 1 ml of ACK lysis buffer. The blood was then stained usingfluorescently-labelled antibodies and the presence of AML or CART cellswas detected using flow cytometry. AML was not detected in mice treatedwith CART33 or CART123.

Disease burden was measured by blasts/ul from retro-orbital bleedings atdifferent time points (FIG. 15).

Survival of mice treated with CART33, CART123 or UTD (p<0.001 wheneither CART33 or CART123 is compared to UTD) was measured (FIG. 16). Theresults presented herein demonstrate CART33 and CART123 produceequivalent eradication of primary AML in vivo.

Hematopoietic Stem Cell Toxicity of CART33 Cells

To determine hematopoietic stem cell toxicity of CART33 cells, humanizedimmune system (HIS) mice were bled retro-orbitally 6-8 weeks afterinjection of human CD34+ cells derived from the fetal liver to confirmengraftment of human cells. Mice were then treated with either CART33 orUTD (1×10⁶ cells each) and followed by serial weekly retro-orbitalbleedings. Mice were then euthanized on day 28 and organs were harvestedand analyzed (FIG. 17).

Analysis of the peripheral blood (via retro-orbital bleeding) by flowcytometry from day 28 at the conclusion of the experiment was performed(FIGS. 18A-18C). Statistical analysis of day 28 peripheral bloodanalysis from mice treated with CART33, UTD or no treatment (n=5) showedCART33 resulted in significant toxicity on monocytes and CD33+ myeloidlineage cells with relative sparing of B cells and platelets (FIG. 19).Bone marrow analysis by flow cytometry on day 28 showed CART33 treatmentresulted in significant reduction in myeloid progenitors (CD34+CD38+)and in hematopoietic stem cells (CD34+CD38-), gated on singlets,huCD45dim, Lineage negative (FIG. 20).

Sections of the femur were taken from the mice on day 28 after treatmentwith UTD T cells or CART33 cells. huCD45 and CD34 staining by IHC wasperformed (FIG. 21). No difference in huCD45 between control T cells andCART33, although both these groups show less huCD45 staining likelyconsistent with an allogeneic human-anti-human effect. There wasspecific reduction of CD34+ cells in mice treated with CART33. Resultsare representative of two experiments.

CART33 and CART123 Produce Equivalent Hematopoietic Toxicity In Vivo

To determine CART33 and CART123 hematopoietic toxicity in vivo, NSGSmice received busulfan i.p. followed by 2×10⁶ T cell depleted bonemarrow cells from a normal donor the following day. Engraftment wasconfirmed by flow cytometric analysis of peripheral blood after 4 weeksand mice were then treated with 1×10(6) autologous T cells, transducedwith CART33, CART123 or UTD. Mice were then followed with retro-orbitalbleeding on day 7 and day 14 and were euthanized for necropsy on day 14(FIG. 22).

Bone marrow analysis was performed by flow cytometry on day 28. CART33and CART123 treatment resulted in significant reduction in myeloidprogenitors (CD34+CD38+) and in hematopoietic stem cells (CD34+CD38−),gated on huCD45dim, Lin−. Results are representative of two experiments(FIG. 23). The results presented herein suggest CART33 and CART123produce equivalent hematopoietic toxicity in vivo.

CD34 enriched BM from patients with MDS was incubated with either UTD,CART33 (IgG4 hinge), CART33 (CD8 hinge), or CART123. There wassignificant reduction in CD45dimCD34+ cells in samples treated withCART33 or CART123 (FIG. 24). The results presented herein demonstrateCART33 and CART123 are cytotoxic myelodysplastic syndrome (MDS) marrowcells.

Additional experiments examining the activity of humanized CART33 aredescribed in Examples 5-6.

Example 2: CAR Constructs

Fully human anti-CD33 single chain variable fragments (scFv) weregenerated and cloned into a lentiviral expression vector with theintracellular CD3zeta chain and the intracellular co-stimulatory domainof 4-1BB and given the names depicted in Table 1 (which is shown in theDetailed Description).

The order in which the VL and VH domains appear in the scFv was varied(i.e., VL-VH, or VH-VL orientation), and where either three or fourcopies of the “G4S” (SEQ ID NO:25) subunit, in which each subunitcomprises the sequence GGGGS (SEQ ID NO:25) (e.g., (G4S)₃ (SEQ ID NO:28)or (G4S)₄(SEQ ID NO:27)), connect the variable domains to create theentirety of the scFv domain, as shown in Table 3.

The sequences of the human scFv fragments (SEQ ID NOS: 39-83, includingthe optional leader sequence) are provided herein in Table 2 (in theDetailed Description). The sequences of human scFv fragments, withoutthe leader sequence, are provided herein in Table 9 (SEQ ID NOS: 255-261for the nucleotide sequences, and SEQ ID NOS: 262-268 for the amino acidsequences) in the Detailed Description.

These clones all contained a Q/K residue change in the signal domain ofthe co-stimulatory domain derived from CD3zeta chain. The CAR scFvfragments were then cloned into lentiviral vectors to create a fulllength CAR construct in a single coding frame, and using the EF1 alphapromoter for expression (SEQ ID NO: 11).

Sequences of CAR constructs and their domain sequences are listed in theDetailed Description. Analysis of the human CAR constructs was conductedas described in Example 4.

Example 3: Humanized CART Sequences

2213 murine anti-CD33 IgG4 nucleotide sequence (SEQ ID NO: 138)GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGCTGCATACGCGTCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGTGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGCTAGCTCTAGAGCCACCATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCCGGATCCAACATCATGCTGACCCAGAGCCCTAGCAGCCTGGCCGTGTCTGCCGGCGAGAAAGTGACCATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGGCCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGAGCGAGGACCTGGCCATCTACTACTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCGGAGGCACCAAGCTGGAAATCAAGAGAGGCGGCGGAGGCTCAGGCGGAGGCGGATCTAGTGGCGGAGGATCTCAGGTGCAGCTGCAGCAGCCAGGCGCCGAGGTCGTGAAACCTGGCGCCTCTGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGGACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAACGACGACATCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGGGAGCCGGCACCACCGTGACCGTGTCATCTTCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATGATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAG TTGG2213 CAR nucleotide sequence (SEQ ID NO: 139)ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCCGGATCCAACATCATGCTGACCCAGAGCCCTAGCAGCCTGGCCGTGTCTGCCGGCGAGAAAGTGACCATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGGCCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGAGCGAGGACCTGGCCATCTACTACTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCGGAGGCACCAAGCTGGAAATCAAGAGAGGCGGCGGAGGCTCAGGCGGAGGCGGATCTAGTGGCGGAGGATCTCAGGTGCAGCTGCAGCAGCCAGGCGCCGAGGTCGTGAAACCTGGCGCCTCTGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGGACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAACGACGACATCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGGGAGCCGGCACCACCGTGACCGTGTCATCTTCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATGATCT ACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC2213 CAR amino acid sequence (SEQ ID NO: 140)MALPVTALLLPLALLLHAARPGSNIMLTQSPSSLAVSAGEKVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQSEDLAIYYCHQYLSSRTFGGGTKLEIKRGGGGSGGGGSSGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFKGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGAGTTVTVSSSGESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR2213 scFv nucleotide sequence (SEQ ID NO: 141)ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCCGGATCCAACATCATGCTGACCCAGAGCCCTAGCAGCCTGGCCGTGTCTGCCGGCGAGAAAGTGACCATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGGCCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGAGCGAGGACCTGGCCATCTACTACTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCGGAGGCACCAAGCTGGAAATCAAGAGAGGCGGCGGAGGCTCAGGCGGAGGCGGATCTAGTGGCGGAGGATCTCAGGTGCAGCTGCAGCAGCCAGGCGCCGAGGTCGTGAAACCTGGCGCCTCTGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGGACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAACGACGACATCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGGGAGCCGGCACCACCGTGACCGTGTCATCT 2213 scFv amino acid sequence (SEQ ID NO: 142)MALPVTALLLPLALLLHAARPGSNIMLTQSPSSLAVSAGEKVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQSEDLAIYYCHQYLSSRTFGGGTKLEIKRGGGGSGGGGSSGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFKGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGAGTTVTVSS 2218 humanized anti-CD33 IgG4H nucleotide sequence(SEQ ID NO: 143)GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGCTGCATACGCGTCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTYTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGTGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGCTAGCTCTAGAGCCACCATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCCGGATCCGAGATCGTGCTGACACAGAGCCCTGGAAGCCTGGCCGTGTCTCCTGGCGAGCGCGTGACAATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGGCCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAGACTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGCCCGAGGACCTGGCCATCTACTACTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGAGAGGCGGCGGAGGCTCTGGCGGAGGCGGATCTAGTGGCGGAGGATCTCAGGTGCAGCTGCAGCAGCCTGGCGCCGAGGTCGTGAAACCTGGCGCCTCTGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGGACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAACGACGACATCAGCTACAACCAGAAGTTCCAGGGCAAGGCCACCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGGGCCAGGGAACCACCGTGACCGTGTCATCTTCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATGATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAG TTGGHumanized my96 (L2H) IgG4H BBz NT (SEQ ID NO: 144)ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCCGGATCCGAGATCGTGCTGACACAGAGCCCTGGAAGCCTGGCCGTGTCTCCTGGCGAGCGCGTGACAATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGGCCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAGACTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGCCCGAGGACCTGGCCATCTACTACTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGAGAGGCGGCGGAGGCTCTGGCGGAGGCGGATCTAGTGGCGGAGGATCTCAGGTGCAGCTGCAGCAGCCTGGCGCCGAGGTCGTGAAACCTGGCGCCTCTGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGGACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAACGACGACATCAGCTACAACCAGAAGTTCCAGGGCAAGGCCACCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGGGCCAGGGAACCACCGTGACCGTGTCATCTTCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATGATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCHumanized my96 (L2H) IgG4H BBz AA (SEQ ID NO: 145)MALPVTALLLPLALLLHAARPGSEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGGGGSGGGGSSGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSSGESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRHumanized my96 (L2H) scFv nt (SEQ ID NO: 146)GAGATCGTGCTGACACAGAGCCCTGGAAGCCTGGCCGTGTCTCCTGGCGAGCGCGTGACAATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGGCCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAGACTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGCCCGAGGACCTGGCCATCTACTACTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGAGAGGCGGCGGAGGCTCTGGCGGAGGCGGATCTAGTGGCGGAGGATCTCAGGTGCAGCTGCAGCAGCCTGGCGCCGAGGTCGTGAAACCTGGCGCCTCTGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGGACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAACGACGACATCAGCTACAACCAGAAGTTCCAGGGCAAGGCCACCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGGGCCAGGGAACCACCGTGACCGTGTCATCTHumanized my96 (L2H) scFv AA (SEQ ID NO: 147)EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGGGGSGGGGSSGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSS

Example 4: Human CAR Constructs

To depict the cell surface expression of scFv's on a Jurkat T cell line,which contains a luciferase reporter driven by an NFAT-regulatedpromoter (termed JNL cells), JNL cells were transduced with a lentiviralvector expressing a cDNA encoding GFP, an scFv that binds to CD19, orcDNAs that encode an scFv, which was raised against hsCD33 (FIG. 27).The cell surface expression of individual scFv's on JNLs was detected byincubating cells with recombinant Fc-tagged hsCD33 followed byincubation with an Fc-specific secondary antibody conjugated tophycoerythrin. It was observed that clones CD33-1, -2, -3, -4, -5, -6,-7, and -9 bound to hsCD33 at appreciable levels relative to JNL cellsexpressing GFP or an scFv that targets CD19, which served as negativecontrols for this assay. It was also observed that CD33-8 lackedappreciable binding to either hsCD33 or protein L (data not shown), abacterial cell surface protein that binds immunoglobulin light chains.The data presented herein demonstrates that clones CD33-1, -2, -3, -4,-5, -6, -7, and -9 encode scFvs that bind hsCD33.

Individual scFv's targeting hsCD33 were evaluated for their ability toelicit NFAT activity in JNL cells (FIG. 28). JNL cells expressing scFv'sagainst hsCD33 were co-cultured with MOLM13 or MOLP8 cell lines, whichexpress hsCD33 or lack hsCD33 cell surface expression, respectively(FIG. 28A; hsCD33, solid green line; isotype control, gray dashed lineand shaded area). FIG. 28B depicts the activation of JNL cellsexpressing an scFv targeting hsCD33 in the presence of MOLM13 (solidlines) or MOLP8 (dashed lines) cells. JNL cells expressing individualscFv's were plated at different effector (i.e. JNL cells) to target(i.e. MOLM13 or MOLP8) ratios and analyzed for the expression ofrelative luciferase units (RLUs) using the Bright-Glo™ Luciferase Assayon the EnVision instrument 24 hours post-incubation. It was observedthat that scFv clones CD33-1, -2, -3, -4, -5, -6, -7, -9, and an scFvbased on the sequence of Mylotarg, a monoclonal antibody that binds tothe human CD33 antigen (CD33-UPenn), were capable of triggeringNFAT-dependent luciferase activity, albeit to varying degrees, in thepresence of MOLM13 relative to MOLP8 or JNL cells expressing GFP.

The activity of scFv's targeting hsCD33 was further assessed indonor-derived primary T cells (FIGS. 29A and 29B). Naïve T cells wereisolated from healthy donor PBMCs by negative selection using standardprotocols and activated using the Dynabeads® Human T-Expander CD3/CD28Invitrogen kit. After 24 hours post-stimulation, T cells were transducedwith lentivirus expressing individual scFv's targeting hsCD33 andexpanded in culture for an additional 9 days. T cell cultures wereanalyzed for their ability to undergo cellular expansion and elicitcytolytic activity in an antigen-dependent and antigen-independentmanner. The expression of scFv's on the cell surface of primary human Tcells transduced with a lentiviral vector that expresses an scFv thattargets hsCD33 is depicted in FIGS. 29A and 29B. The expression ofscFv's was detected by incubating cells with recombinant Fc-taggedhsCD33 and an Fc-specific secondary antibody conjugated to phycoerythrinas described in FIG. 27. Consistent with the aforementioned findings inFIG. 27, scFv clones CD33-1, -2, -3, -4, -5, -6, -7, and -9 expressed inprimary T cells bound to hsCD33 at appreciable levels relative to Tcells expressing GFP, which lack the ability to bind hsCD33. Thus, scFvclones CD33-1, -2, -3, -4, -5, -6, -7, and -9 can be used to engineerprimary T cells to target the human CD33 antigen.

T cells were labeled with CFSE and co-cultured in the presence of MOLM13(FIG. 30A, solid black bar), MOLP8 (FIG. 30A, open white bar), orcultured alone (FIG. 30A, hatched bar) to assess the proliferativecapacity of UTD primary T cells or cells expressing scFv's targetinghsCD33. It was observed that T cells expressing scFv clones CD33-2, -3,-4, -5, -6, and -9 were able to expand in an antigen-dependent manner,as evidenced by an increase in the absolute number of T cells in thepresence of CD33-expressing MOLM13 cells in comparison to UTD orCD33-negative MOLP8 cells (FIG. 30A). In addition, antigen driven celldivision in primary T cells was assessed by measuring the medianfluorescence intensity (MFI) of CFSE-labeled T cells expressing scFvclones CD33-2, -3, -4, -5, -6, and -9 co-cultured with MOLM13, MOLP8cells, or alone (FIG. 30B). Consistent with the aforementioned increasein T cell expansion (FIG. 30A), diminished CFSE levels in T cellsco-cultured with MOLM13 in comparison to UTD or MOLP8 cells was observed(FIG. 30B). Upon gating on T cells that were either scFv⁺ or scFv⁻, itwas found that scFv-expressing T cells had diminished CFSE levelsrelative to their scFv⁻ counterparts when co-cultured with MOLM13.However, CFSE levels were similar between scFv⁺ or scFv⁻ T cellsco-cultured on MOLP8 cells (FIG. 30B), which is indicative of increasedproliferation in response to antigen recognition. The results presentedherein are also consistent with the ability of scFv clones CD33-2, -3,-4, -5, -6, and -9 to elicit scFv-dependent activation of JNL cells inthe presence of CD33 (FIG. 28B). Thus, scFv clones CD33-2, -3, -4, -5,-6, and -9 can be used to activate the proliferation of primary T cellsin a CD33-dependent manner.

Additionally, CART-CD33 T cells were tested for their ability toproliferate in response to exposure to antigen on target cells. Targetcells included PL-21, HL60, and Molp8 cells. On the day of assay (Day0), target cells were counted and transferred to a 50 ml tube in 6 mL ofT cell media at 3e6 cells/ml. Target cells were irradiated on ice at10,000 rad. After irradiation, target cells were washed twice in T cellmedia, counted, and resuspended to 5e5 cells/ml in T cell media on ice.Frozen transduced T cells were thawed, washed in 10 mL complete T cellmedia, spun at 300 g for 10 min, and resuspended gently in 3 mL ofcomplete T cell media at RT. T cells were then counted in a cellometerand resuspended to 2.5e6/mL in 10 mL of media. In a 96 well U-bottomplate, 25,000 irradiated target cells and 25,000 transduced CAR T cells(1:1 ratio) were combined in duplicate wells. In a separate well, 75,000anti-CD3/CD28 beads were added in 100 μl of medium to 25,000 transducedT cells to create a 1:3 cells-to-beads ratio as positive control; inanother well, 100 μl of medium was added to 25,000 transduced T cellsalone as media-only control. Cells were incubated for 4 days at 37° C.,5% CO₂. On day 4, cells were harvested and duplicates were combined bypipetting and transferring into the same well on the U-bottom plate forstaining for FACS of CD4, CD8, and CAR using protein L or recombinanthuman CD33 protein. After staining, cells were resuspended in 120 μlMACS+0.5% BSA buffer and 20 μl/well countbright beads were added to eachwell. Proliferation was measured as the number of FACS positive cellsdetected in the period of time used to count 2500 beads (FIG. 47). Thetested CART-CD33 T cells (e.g., derived from scFv clones CD33-1, -2, -4,-5, -6, -7, -9, and humanized My96 (termed “Upenn”) proliferated to agreater extent than untransduced cells or CART-CD19 T cells when exposedto HL-60 target cells. The CART-CD33 T cells (e.g., derived from scFvclones CD33-1, -2, -4, -5, -6, -7, -9, and humanized My96 (termed Upenn)proliferated to a greater extent than untransduced cells and to an aboutequal or greater extent than CART-CD19 T cells when exposed to PL21target cells. The CART-CD33 T cells proliferated to about the sameextent or slightly greater extent than untransduced cells or CART-CD19 Tcells when exposed to MOLP8 cells which do not express CD33 target.

To assess cytolytic activity, 25,000 MOLM13 cells were plated withprimary T cells expressing individual scFv's at different effector (i.e.T cell) to target (i.e. MOLM13) ratios and analyzed for the extent ofMOLM13 killing by enumerating the absolute number of CFSE-labeled MOLM13cells after 4 days in culture (FIG. 31). It was observed that scFvclones CD33-2, -3, -4, -5, -6, and -9 were capable of inducing targetcell lysis, albeit to varying degrees. Thus, scFv clones CD33-2, -3, -4,-5, -6, and -9 can be used to engineer primary T cells to directlytarget and kill CD33-expressing target cells.

T cell killing was directed toward CD33-expressing PL21 and HL-60 acutemyelogenous leukemia cell lines stably expressing luciferase. Non-CD33expressing U87 cells were used as a control and untransduced T cells(UTD) were used to determine non-specific background killing levels. Thecytolytic activities of CART-CD33 were measured as a titration ofeffector:target cell ratios of 10:1 and 2-fold downward dilutions of Tcells where effectors were defined as T cells expressing the anti-CD33chimeric receptor. Assays were initiated by mixing an appropriate numberof T cells with a constant number of targets cells. After 20 hoursluciferase signal was measured using the Bright-Glo™ Luciferase Assay onthe EnVision instrument. Comparing these killing curves, titrating theamount of effector cells shows that those cells expressing CD33 weredestroyed (FIGS. 48A, 48B, and 48C). T cells from the same donor thatwere transduced with any of the human scFv bearing CAR-CD33 cells wereable to kill selectively CD33+ targets, although differences in activitywere noted that could translate into differences in clinical activity.

CART-CD33 cells were tested for their ability to produce cytokine inresponse to antigen. Cells were thawed and allowed to recover overnight.Untransduced T cells (UTD) were used as a non-specific control forbackground T cell effects. The T cells were directed towards HL-60,PL21, or MOLP8 cells. The assay tested an effector:target ratio of 2.5:1where effectors were defined as T cells expressing the anti-CD33 CAR.The assay was run 16 hours after mixing of the cells, when the media isremoved for analysis of cytokines IFN-gamma, TNF-alpha, and IL-2 usingthe CBA-Flex kit for human cytokine detection. When CART-CD33 T cellswere cultured with cancer cells endogenously expressing CD33, allCD33-CARTs produced cytokines in response to target-expressing cells(FIG. 49). The difference in reactivity of the various CD33-CART clonestoward low CD33-expressing target cells may translate to better clinicalefficacy of CART cells transduced with these constructs.

Finally, scFv cross-reactivity to cynomolgus CD33 (cyCD33) was assessedby flow cytometry as depicted in FIG. 32. JNL cells transduced with alentiviral vector expressing scFv's raised against hsCD33 were incubatedwith either recombinant Fc-tagged hsCD33 (red line) or cyCD33 (blueline) followed by incubation with an Fc-specific secondary antibodyconjugated to phycoerythrin. While scFv clones CD33-1, -2, -3, -4, -5,-6, -7, -9, and CD33-UPenn were capable of binding hsCD33, we note thatonly scFv clone CD33-5 appeared to be cross-reactive to both hsCD33 andcyCD33 (FIG. 32).

Example 5: Characterization of RNA-Electroporated CART33 Cells

Comparison of CAR33 Expression in T Cells Lentivirally Transduced or RNAElectroporated

An mRNA anti-CD33 CAR comprising a humanized anti-CD33 scFv and an IgG4hinge, e.g., SEQ ID NO: 145, was generated by in vitro transcription. Tcells from a normal donor was isolated and electroporated with the CAR33mRNA. After electroporation, cells were stimulated with CD3/CD28 beadsand expanded in culture. CAR33 expression was measured and quantified byflow cytometry at 8 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6days, or 7 days after electroporation (FIG. 33A). The results from thisexperiment demonstrate that RNA electroporation of CAR33 results inCAR33 expression in donor T cells for at least 7 days. Expression washighest (e.g., greater than 60%) through day 4 after electroporation(FIG. 33B). CAR33 expression decreased from days 5-7 afterelectroporation.

CAR33 expression from RNA electroporation was compared to stable CAR33expression from lentiviral transduction. Donor T cells were transducedwith a lentiviral construct comprising a CAR33, such as the vector ofFIG. 26, using standard methods. CAR33 expression was measured by flowcytometry analysis and quantified by mean fluorescence intensity (MFI).CAR33 expression was assessed at 8 hours, 1 day, 2 days, 3 days, and 4days post electroporation or transduction for lentivirally-transducedCART33 cells (FIG. 34A) and for RNA-transfected CART33 cells (FIG. 34B).The grey peaks at the left side of the histograms of FIG. 34 representslack of CAR33 expression, while increased CAR33 expression increasestowards the right side of the graphs. Lentiviral transduction results instable expression of CAR33 in T cells, wherein the CAR33 expressionlevel remains unchanged from 8 hours to 4 days after transduction. Incontrast, RNA electroporation results in transient expression of CAR33in T cells. Specifically, CAR33 expression is highest at 8 hours to 1day after electroporation, and CAR33 expression decreases from 2 to 4days after electroporation.

In Vitro Cytotoxic Activity

To assess the specific killing activity of RNA-electroporated andlentiviral-transduced CART33 cells, CART33 cells were incubated withCD33-expressing target cells, such as the acute myeloid leukemia cellline MOLM14, or CD33-negative control cells, such as the mantle celllymphoma cell line JEKO at varying effector (CART33 cells) to target(CD33 positive or negative cells) ratios for 24 hours. Effector totarget ratios ranged from 0:1, 0.125:1, 0.25:1, 0.5:1, 1:1, and 2:1. Theexperiment was repeated at different time points after 1 day, 2 days, 3days, and 4 days electroporation/transduction of the T cells. One dayafter electroporation, RNA-electroporated CART33 cells exhibitedspecific killing starting at the E:T ratio of 0.125:1 (FIG. 35A).Specific killing of CAR33-positive MOLM14 cells was observed at 2 (FIG.35B), 3 (FIG. 35C) and 4 (FIG. 35D) days post electroporation of theCART33 cells, however the E:T ratios for specific killing increased overtime, indicating the transient nature of the specific killing.Lentivirally transduced CART33 cells exhibited potent specific killingactivity from 1 to 4 days even at the lower E:T ratios.

When the results were displayed in a different way, with the specificactivity of one particular E:T ratio compared between 1 to 4 days afterelectroporation, the data shows that the specific killing ofRNA-electroporated CART33 cells decreased over time afterelectroporation when E:T ratios were at 2:1 (FIG. 36A) or 1:1 (FIG.36B). These results demonstrate the transient nature of the specifickilling of RNA-electroporated CART33 cells. In contrast, lentivirallytransduced CART33 cells exhibited stable levels of specific killing fromdays 1-4 after transduction.

Example 6: Humanized CART33 Exhibit Potent Preclinical Activity AgainstHuman AML and MDS

A second generation CAR was constructed from the anti-CD33 scFv ofGemtuzumab ozogamicin (an immunoconjugate targeting CD33) with 4-1BB andCD3 zeta signaling domains (described in Example 3; and SEQ ID NO: 145).Here, the preclinical activity of the second generation CAR33 isdescribed, and compared to previously developed CAR targeting CD123(CAR123). The results show that CART33 was able to eradicate human acutemyeloid lymphoma and myelodysplastic syndrome CD34+ cells, whileresulting in significant myelotoxicity in mouse xenografts. Thus,transiently expressed mRNA modified CART33 were also generated to beused in future studies and clinical trials.

The following materials and methods were used in this example:

Generation of CART Cells

The pTRPE anti-CD33-41BB-CD3 zeta (CAR33) plasmid DNA was generated bycloning the light to heavy chain orientations of the humanizedanti-human CD33 scFv derived from gemtuzumab ozogamicin (clone my96)into the previously described murine CART19 plasmid vector. 25 Normaldonor T cells were positively selected from leukapheresis packs usinganti CD4 and anti CD8 microbeads (Miltenyi), mixed together at 1:1 ratioand expanded in vitro using anti-CD3/CD28 Dynabeads (Invitrogen, addedon the first day of culture) with low dose IL-2. T cells were transducedwith lentiviral supernatant from 293T cells transfected with pTRPEmy96-CD33-41BB-CD3zeta plasmid on day following stimulation at amultiplicity of infection (MOI) of 3. The antiCD3/CD28 Dynabeads wereremoved on day 6 and T cells were expanded in culture in T cell media(X-vivo 15 media, supplemented with human serum 5%, penicillin,streptomycin and glutamax) for up to 15 days and then cryopreserved forfuture experiments. Prior to all experiments, T cells were thawed andrested overnight at 37 degrees. Production of CART123 cells waspreviously described (Gill et al., 2014, Blood, 123:2343-54).

Cells

The MOLM14 cell line was obtained from the ATCC and maintained in R10media (RPMI media supplemented with 10% fetal calf serum, penicillin,and streptomycin). MOLM14-luciferase-GFP cells were used in someexperiments. De-identified primary human AML and MDS bone marrowspecimens were obtained from the University of Pennsylvania Stem Celland Xenograft Core facility. For all functional studies, AML cells werethawed at least 12 hours before analysis and rested overnight in 37degrees. MDS bone marrow samples were enriched for CD34+ cells bypositive selection using MACSQuant columns (Miltenyi).

Flow Cytometry Analysis

Anti-human antibodies were purchased from Biolegend, eBioscience, orBecton Dickinson. Cells were isolated from in vitro culture or fromanimals, washed once in PBS supplemented with 2% fetal calf serum, andstained on ice after blockade of Fc receptors. For cell numberquantitation, Countbright beads were used according to themanufacturer's instructions (Invitrogen). In all analyses, thepopulation of interest was gated based on forward vs. side scattercharacteristics followed by singlet gating, and live cells were gatedusing Live Dead Aqua (Invitrogen). Surface expression of anti-CD33 CARwas detected by staining with an Alexa Fluor 647-conjugated goatantimouse F(ab′)2 antibody from Jackson Immunoresearch. Flow cytometrywas performed on a four-laser Fortessa analyzer (Becton-Dickinson).

T Cell Function Assays

1. T Cell Degranulation Assays.

Degranulation assays were performed as previously described.26 T cellswere incubated with target cells at a 1:5 ratio. CD107a, CD28, CD49d andmonensin were added at the time of incubation. After 4 hours, cells wereharvested and stained for CAR expression, CD3 and Live Dead staining.Cells were fixed and permeabilized and intracellular cytokine stainingwas then performed.

2. Proliferation Assays:

T cells were washed and resuspended at 1×107/ml in 100 ul of PBS andstained with 100 ul of CFSE 2.5 μM (Life Technologies) for 5 minutes at37 Celsius. The reaction was then quenched with cold R10, and the cellswere washed three times. Targets were irradiated at a dose of 100 Gy. Tcells were incubated at a 1:1 ratio with irradiated target cells for 120hours. Cells were then harvested, stained for CD3, CAR and Live Deadaqua, and Countbright beads (Invitrogen) were added prior to flowcytometric analysis.

3. Cytotoxicity Assays:

Luciferase+ MOLM14 cells or CF SE labelled primary AML samples were usedfor cytotoxicity assay as previously described (Cao et al., 2010,Cytometry A, 77:534-545). In brief, targets were incubated at theindicated ratios with effector T cells for 4 or 16 hours. Killing wascalculated either by bioluminescence imaging on a Xenogen IVIS-200Spectrum camera or by flow cytometry. For the latter, cells wereharvested, and Countbright beads and 7-AAD were added prior to analysis.Residual live target cells were CFSE+ 7-AAD−. For MDS, T cells wereincubated with CD34-enriched bone marrow at 1:1 ratio for 4 or 24 hoursas indicated and cytotoxicity was then measured by flow cytometry or byfluorescence in situ hybridization (using probe for a specificchromosomal abnormality in the MDS sample).

4. Cytokine Measurements:

Effector and target cells were incubated at a 1:1 ratio in T cell mediafor 24 or 72 hours as indicated. Supernatant was harvested and analyzedby 30-plex Luminex array according to the manufacturer's protocol(Invitrogen).

In Vivo Experiments

NOD-SCID-γ chain−/− (NSG) and NSG mice transgenic for human IL-3, stemcell factor, and GM-CSF (NSG-S) originally obtained from Jackson Labs.All experiments were performed on protocols approved by theInstitutional Animal Care and Use Committee of the University ofPennsylvania. Schemas of the utilized xenograft models are discussed indetails in the relevant figures and the results section. Cells (MOLM 14,primary cells or T cells) were injected in 200 μl of PBS at theindicated concentration into the tail veins of mice. Bioluminescentimaging was performed using a Xenogen IVIS-200 Spectrum camera.Humanized immune system (HIS) mice were created by injection of fetalliver CD34+ cells into newborn NSG mice and were used at approximately 8weeks of age.

Generation of mRNA-Modified CART33

The CAR construct from the pTRPE anti-CD33-41BB-CD3z plasmid wassubcloned into the pDA vector 28 as previously published. In-vitrotranscription was performed using mMESSAGE mMachine®T7 ULTRAtranscription kit (Ambion). The RNA was purified using RNeasy Mini Kit(Qiagen). RNA-CAR33 was electroporated into T cells as previouslydescribed. Electroporation was done using an ECM830 Electro Square WavePorator (Harvard Apparatus BTX).

Histology and Immunohistochemistry

Formalin-fixed, paraffin-embedded sections from mouse femurs werestained with hematoxylin and eosin, counterstained with mAbs to humanCD45 and human CD34 and acquired with a Nikon microscope.

Results:

CD33 as a Target in AML and CART33 Constructs

To verify the clinical relevance of CD33 as a target for immunotherapyin AML, the level of expression of CD33 in AML was first assessed andwas found to be expressed on the majority of AML blasts in almost allprimary AML samples (FIG. 1) as well as in bone marrows from MDSpatients (FIGS. 2A, 2B, and 2C). To assess the potential off-targettoxicity of CART33, tissue immunohistochemistry was performed on 30normal tissues stained with anti-CD33 antibody (LSBio). CD33 wasexpressed on myeloid lineages in the bone marrow and on residentmacrophages in the liver lung and kidneys (FIG. 21). To test theefficacy of CAR33, four constructs were designed derived from the murineand humanized scFv from Gemtuzumab Ozogamicin clone my96. Two constructsutilized IgG4 hinge and two constructs used CD8 hinge (FIG. 3).

CART33 Show Potent In Vitro Activity Against AML Cell Lines, Primary AMLSamples and MDS

The activity of the four different CART33 constructs was tested in vitroand compared that to CART123 (Gill et al, 2014, Blood, 123:2343-2354).Humanized CART33 was consistently superior to murine CART33 (seeExample 1) and therefore all subsequent studies were performed on thetwo humanized CAR33 constructs. MOLM14 cell line was employed as a modeltumor (MOLM14 expresses CD33 and CD123). Incubation of CART33 withMOLM14 resulted in significant degranulation (FIG. 37A), potentcytotoxicity at low effector: target ratios (FIG. 37B), extensiveproliferation (FIGS. 37C and 37D) and robust cytokine production (FIGS.38A, 38B, 38C, and 39) that was significantly higher than incubationwith the control T cell leukemia cell line Jurkat. The majority ofCART33 produced two or more cytokines per cell after incubation withMOLM14, in a similar pattern to potent nonspecific stimulation withPMA/Ionomycin (FIGS. 38A and 38B). This function has been associatedwith superior in vivo activity (Carpenito et al., 2009, Proc Natl AcadSci USA, 106:3360-3365). Furthermore, CART33 cells produced morecytokines than CART123 cells (FIG. 38C) and also resulted in significantin vitro activity against primary AML samples. CART33 with IgG4 hingeresulted in superior cytotoxicity compared with CART33 with CD8 hinge(FIGS. 8, 9, 10 and 40).

In vitro activity of CART33 in MDS was also examined. Bone marrowsamples from MDS patients were CD34 enriched (˜85% purity) and thenincubated with CART33, CART123 or untransduced control T cells (UTD)cells at E:T ratio of 1:5 for 4 hours, in the presence of CD49d, CD28co-stimulation and momensin. CD107a degranulation was then measured byflow cytometry and percentage of degranulation was quantified. FIGS. 41Band 41C shows specific killing of the MDS clone having 5q deletion. CD34enriched bone marrow sample from a patient with MDS and 5q deletion wasincubated with CART33, UTD cells or with no treatment at 1:1 E:T ratiofor 4 hours. Sample was then harvested and FISH for 5q− was performed(FIG. 41B). CART33 exhibited significant in vitro activity in MDS. Thiswas evident by specific degranulation of CART33 in response to CD34enriched MDS samples (FIG. 41A), specific killing after a 24-hourincubation of CART33 with CD34-enriched MDS samples (1:1 effector:targetratio, FIG. 24), and the specific reduction of the malignant clone(measured by FISH) after 4 hours of incubation (FIGS. 41B and 41C).

Results from the in vitro assays described above are summarized in theTable below.

TABLE 5 In vitro activity of CART33, compared to CART123 and controluntransduced T cells T cell effector function CART33* UTD %Degranulation (4 hr incubation with   98% 1.3% MOLM14) % Specific Lysis(E:T 0.625:1) 65.7%  30% CFSE based proliferation (5-day incubation91.6%   3% with MOLM14) Cytokine IL6 22.65 5.73 production, 24-hourGM-CSF 13248 16.9 incubation with MIP-1b 8180 9.15 MOLM14 INF-g 249890.35 (median, IL-2 9300 0.39 pg/ml) *All p values are <0.05 whencompared to UTD

CART33 Result in Reduction of Leukemia Burden and Survival Advantages inMOLM14 Engrafted Xenografts

To test the in-vivo activity of CART33, NSG mice were injected withluciferase+ MOLM14 (FIG. 11). After confirmation of engraftment bybioluminescence imaging mice received a single injection of CART33, orcontrol untransduced T cells (UTD) at different dose levels. Mice werethen followed with serial imaging and disease burden was quantifiedusing bioluminescence. Mice treated with control T cells succumbedquickly to disease, while mice treated with CART33 showed significantreduction of the disease and a survival advantage (FIGS. 42A, 42B, and42C). Furthermore, dose response was examined in CART33 treated mice. Asshown in the schematic in FIG. 43A, NOD-SCID-gamma chain knockout (NSG)mice were injected with the AML cell line MOLM14 1×10⁶ I.V. and imagedfor engraftment after 6 days. Mice were treated with CART33 5×10⁶,CART33 2×10⁶, CART33 1×10⁶, or control untransduced T cells 5×10⁶. Themice were followed with serial weekly imaging to assess the burden ofAML. A dose dependent response (FIGS. 43A and 43B) was observed inCART33 treated mice and superior anti-leukemic activity of CART33 withIgG4 hinge compared to CAR33 with CD8 hinge (FIG. 44). For allsubsequent experiments, only CART33 with an IgG4 hinge were used.

CART33 Result in Eradication of Leukemia in Primary AML Xenografts andin Long Term Disease Free Survival

Primary leukemia cells are likely more clonally heterogeneous thanlong-term propagated cell lines and are more representative of the humandisease. Therefore, the efficacy of CART33 in primary AML xenografts wasevaluated. NSG-S mice were injected with primary AML samples expressingCD33 and CD123. Disease burden was quantified in the peripheral bloodand by survival analysis (FIG. 13). Engraftment was defined as >1%circulating huCD45+ cells and was typically achieved 2-4 weeks afterinjection of the leukemic cells. These mice were then treated with asingle injection of CART33, CART123 or UTD cells (1×10⁵ via tail veininjection). Leukemia was eradicated within 4 weeks of CART33 or CART123injection (FIGS. 14A-14C and 15) and long term disease free survival wasdemonstrated (FIG. 16).

CART33 Result in Expected Hematopoietic Stem Cells and MyeloidProgenitors Toxicity

Since CD33 is known to be expressed on myeloid progenitor, albeit tolower levels compared to leukemic cells, the impact of CART33 on normalhematopoiesis was investigated. Two different models were used to assesshematopoietic toxicity of CART33. Humanized immune system (HIS) micepostnatally engrafted with human fetal CD34+ cells were bled to confirmengraftment and then were treated with CART33, CART123 or untransduced Tcells (FIG. 17). Mice were bled weekly for 4 weeks. Mice were theneuthanized and bone marrow and spleen from these mice were collected foranalysis. As expected based on CD33 expression on myeloid lineage, thesemice developed reduction in peripheral blood myeloid cells includingmonocytes compared to mice treated with untransduced T cells (FIG. 22).Analysis of the bone marrow 4 weeks after treatment showed reduction ofthe CD34+CD38-hematopoietic stem cell and the CD34+CD38+ myeloidprogenitors by flow cytometry (FIG. 20) or immunohistochemistry (FIG.21). The HIS model is biased toward B-cell lineage and so a second modelthat is more myeloid biased was employed. Here, bone marrow from normaladult donors was T cell depleted and injected into busulfan-conditionedNSG-S mice. Autologous CART33, CART123, or UTD were generated bytransducing peripheral blood T cells from the marrow donor with therelevant lentivirus. After confirming engraftment of these mice, theywere treated with autologous CART33, CART123, or UTD and followed withweekly retro-orbital bleedings for a total of 4 weeks (FIG. 22). Micewere then euthanized and tissues were harvested and analyzed. Similar tothe HIS xenografts, we observed reduction in peripheral blood myeloidcells and monocytes and in the CD34+ marrow compartments.

Transient RNA-Modified “Biodegradable” CART33 Result in Potent butTransient Anti-Leukemia Activity

Since CD33 is expressed on normal hematopoietic cells and tissueresident macrophages (FIGS. 1 and 2), it is important to validate asafety mechanism prior to clinical application. Therefore, RNA-modifiedCART33 was developed. Electroporation of T cells with RNA-modifiedCAR-33 resulted in high level expression of CAR that graduallydiminished over seven days (FIGS. 33A, 33B, and 34, and as described inExample 9). When compared to lentivirally transduced CART33 (LV-CART33),RNA-modified CART33 has similar, but transient in-vitro activity.Incubation of MOLM14 with RNA-CART33 resulted in specific cytotoxicitycomparable to LV-CART33 at E:T ratios of 1:1 and 2:1 and that decreasedwith time post electroporation (FIGS. 35A-35D, and as described inExample 9).

The combination of RNA-CART33 with chemotherapy in vivo was tested. NSGmice engrafted with MOLM14 were treated with either the combination ofcyclophosphamide (60 mg/kg I.P) three doses plus RNA-CART33 orcyclophosphamide plus UTD. 10×10⁶ T cells were given IV in three doses(FIG. 45). Specifically, NSG mice were injected with MOLM14-luc (1×10⁶I.V) and imaged to confirmed engraftment four days later. Mice were thenrandomized to receive either RNA-CART33 with cyclophosphamide oruntransduced T cells with cyclophosphamide (60 mg/kg I.P). T cell weregiven at a dose if 10×10⁶ I.V in three different doses, on days 5,9,16.Cyclophosphamide was given on days 8 and 14. The combination ofcyclophosphamide and RNA modified CART33 resulted in improved leukemiacontrol compared to control mice.

Discussion

In this example, the preclinical activity and safety of CART33 in AMLwas detected and transiently expressed CART33 was developed andvalidated as a way to avoid toxicity when used in patients withrefractory AML. This is the first extensive preclinical report of theactivity of CART33 that includes comprehensive survival and toxicitydata incorporating multiple mouse models as well as the anti-leukemicactivity of RNA-modified CART33. CART33 exhibited potent effectorfunctions against AML cell line and primary AML samples, includingspecific killing at low E:T ratios, degranulation, profoundproliferation and robust cytokine production and were also active inreducing an MDS clone after only 4 hours of incubation at 1:1 ratio.

Treatment with CART33 also resulted in eradication of AML and survivaladvantages in both MOLM14 and primary AML xenografts after a singleinfusion. The expected myeloid hematopoietic toxicity with CART33 wasobserved in two different humanized mouse models. Because of thepotential myelotoxicity and concerns for CD33 expression on residenttissue macrophages, an RNA-modified CART33 was developed and potent, buttransient in vitro activity was shown. An anti-leukemic effect was alsoshown by combining RNA-CART33 and chemotherapy.

The experiments described herein and in Example 1 also showed that usingan IgG4 hinge was superior to using a CD8 hinge with CART33. The IgG4molecule is significantly different from CD8 molecule. It contains threetimes more amino acids which could result in a more flexible hinge.

The hematopoietic toxicity and reduction in myeloid progenitor as wellas peripheral blood cytopenia observed with CART33 in these preclinicalstudies is expected based on CD33 expression on leukemic as well asmyeloid progenitors.

The potential for off target toxicity with the use of CART33 mandatesthe incorporation of transiently expressed, rather than permanent, antiCD33 therapy in clinical trials. RNA-modified CARs have been utilized inpreclinical models (Barrett et al., 2013, Hum Gene Ther, 24:717-727; andBarrett et al., 2011, Hum Gene Ther, 22:1575-1586), and a phase I trialof RNA-modified anti-mesothelin CAR T cells in patients with solid tumorhas showed that this approach is safe and feasible (Beatty et al., 2014,Cancer Immunol Res, 2:112-120). Hence, RNA-modified CART33 was developedas a way of transiently expressed “biodegradable” CARs to mitigate thepossible off-target toxicity of CART33.

Since recent larger clinical trials showed survival advantages when GOwas combined with chemotherapy in low and intermediate risk disease(Hills et al., 2014, Lancet Oncol., 15:986-996), the efficacy of thecombination of multiple infusions of RNA-modified CART33 withchemotherapy in AML, mouse xenografts was tested (FIG. 45). Thiscombination resulted in deeper and longer responses and in survivaladvantages for these mice. These observations highlight several fewpotential translational applications for RNA-modified CART33. This canbe used alone or in combination with chemotherapy in patients withrelapsed refractory AML who are unable to undergo allogeneic stem celltransplantation to render them transplant eligible. In addition,RNA-modified CART33 can be incorporated in conditioning regimens priorto allogeneic stem cell transplantation. Once the safety and feasibilityof this approach have been demonstrated in patients, future strategiescould include lentiviral CART33 with an “off” switch. Furthermore, thesefindings that both CART33 and CART123 are effective against AML open upnew therapeutic horizons in combinatorial targeted cellular therapy.

Example 7: Use of Chimeric Antigen Receptor T Cell Therapy AgainstMyeloid-Derived Suppressor Cells (MDSC) in Cancer

Recent data show that the myelodysplastic syndrome (MDS) marrow milieucontains a population of CD33-expressing myeloid-derived suppressorcells (MDSC) that play an important role in pathogenesis of MDS bysecreting cytokines that promote ineffective hematopoeisis as well asimmunosuppression (Chen et al. J. Clin. Investig. 23(2013): 21-223).Chen et al. described that MDSC play a role in the induction ofmyelodysplasia by a CD33-S100A9 interaction. This example describesexperiments to determine whether MDSC can be targeted using anti-CD33CAR T cell therapy. The results described herein show that both theabnormal MDS clones and their supportive MDSC population may be targetedsimultaneously with a single anti-CD33 CAR T cell product.

MDSC were phenotyped in 4 MDS and 3 normal marrow specimens (FIGS. 50A,50B, and 50C). For the phenotyping, flow cytometry was performed usingthe following gating strategy: MDSCs were defined as lineage negative(LIN−), HLA-DR negative, CD33 positive cells in MDS bone marrow samples(FIG. 50A). MDSCs were more abundant in the dysplastic bone marrow(MDS-BM) compared to the normal bone marrow (ND-BM) (FIG. 50B). Inaddition, the CD33 mean fluorescence intensity (MFI) in the MDSCpopulation was comparable to the CD33 MFI in the malignant MDSpopulation and was significantly higher than the CD33 MFI in thehuCD45+LIN− population from ND-BM (FIG. 50C). Thus, the CD33 expressionin MDSCs was comparable to its expression in malignant MDS populationand was significantly higher than its expression in normal bone marrows.The ability of CART33 to mount a response to MDS blasts and to MDSC fromMDS marrow was assessed by quantifying the extent of CD107adegranulation and cytokine production from CART33 cells. CART33 wereincubated for four hours with negative control (Jurkat cells), positivecontrols (PMA and ionomycin), sorted MDS CD34+, or sorted MDSC from MDSmarrow. Using the the CAR33-UPenn construct (derived from the anti-CD33scFv of gemtuzumab ozogamicin), it was found that MDS CD34 cells andMDSC induced broadly equivalent responses in CART33 (FIGS. 51A and 51B).These results indicate that CART-33 having lower affinity may havedifferential activity against MDSC.

MDSC also play a role in the resistance to immune attack of multipleother malignancies, including chronic lymphocytic leukemia (CLL) (wherethey are known as nurse-like cells), as well as solid malignancies suchas ovarian cancer, breast and colon cancer (Di Mitri et al. Nature515.7525(2014):134-137; Gabrilovich et al. Nat. Reviews Immunol.12.4(2012):253-68; and Kim et al. Proc. Acad. Sci. U.S.A.111.32(2014):1-6). These results demonstrate that MDSC can be targetedwith CART33 either singly or in combination with other immunotherapies.

Example 8: Low Dose RAD001 Stimulates CART Proliferation in a CellCulture Model

The effect of low doses of RAD001 on CAR T cell proliferation in vitrowas evaluated by co-culturing CART-expressing cells with target cells inthe presence of different concentrations of RAD001.

Materials and Methods

Generation of CAR-Transduced T Cells A humanized, anti-human CD19 CAR(huCART19) lentiviral transfer vector was used to produce the genomicmaterial packaged into VSVg pseudotyped lentiviral particles. The aminoacid and nucleotide sequence of the humanized anti-human CD19 CAR(huCART19) is CAR 1, ID 104875 described in PCT publication,WO2014/153270, filed Mar. 15, 2014, and is designated SEQ ID NOs. 85 and31 therein.

Lentiviral transfer vector DNA is mixed with the three packagingcomponents VSVg env, gag/pol and rev in combination with lipofectaminereagent to transfect Lenti-X 293T cells. Medium is changed after 24h and30h thereafter, the virus-containing media is collected, filtered andstored at −80° C. CARTs are generated by transduction of fresh or frozennaïve T cells obtained by negative magnetic selection of healthy donorblood or leukopak. T cells are activated by incubation withanti-CD3/anti-CD28 beads for 24h, after which viral supernatant orconcentrated virus (MOI=2 or 10, respectively) is added to the cultures.The modified T cells are allowed to expand for about 10 days. Thepercentage of cells transduced (expressing the CARs on the cell surface)and the level of CAR expression (relative fluorescence intensity, GeoMean) are determined by flow cytometric analysis between days 7 and 9.The combination of slowing growth rate and T cell size approaching ˜350fL determines the state for T cells to be cryopreserved for lateranalysis.

Evaluating Proliferation of CARTs

To evaluate the functionality of CARTs, the T cells are thawed andcounted, and viability is assessed by Cellometer. The number ofCAR-positive cells in each culture is normalized using non-transduced Tcells (UTD). The impact of RAD001 on CARTs was tested in titrations withRAD001, starting at 50 nM. The target cell line used in all co-cultureexperiments is Nalm-6, a human pre-B cell acute lymphoblastic leukemia(ALL) cell line expressing CD19 and transduced to express luciferase.

For measuring the proliferation of CARTs, T cells are cultured withtarget cells at a ratio of 1:1. The assay is run for 4 days, when cellsare stained for CD3, CD4, CD8 and CAR expression. The number of T cellsis assessed by flow cytometry using counting beads as reference.

Results

The proliferative capacity of CART cells was tested in a 4 dayco-culture assay. The number of CAR-positive CD3-positive T cells (darkbars) and total CD3-positive T cells (light bars) was assessed afterculturing the CAR-transduced and non-transduced T cells with Nalm-6(FIG. 54). huCART19 cells expanded when cultured in the presence of lessthan 0.016 nM of RAD001, and to a lesser extent at higher concentrationsof the compound. Importantly, both at 0.0032 and 0.016 nM RAD001 theproliferation was higher than observed without the addition of RAD001.The non-transduced T cells (UTD) did not show detectable expansion.

Example 9: Low Dose RAD001 Stimulates CART Expansion In Vivo

This example evaluates the ability of huCAR19 cells to proliferate invivo with different concentrations of RAD001.

Materials and Methods:

NALM6-luc cells: The NALM6 human acute lymphoblastic leukemia (ALL) cellline was developed from the peripheral blood of a patient with relapsedALL. The cells were then tagged with firefly luciferase. Thesesuspension cells grow in RPMI supplemented with 10% heat inactivatedfetal bovine serum.

Mice: 6 week old NSG (NOD.Cg-Prkdc^(scid)Il2rg^(tmIWjl)/SzJ) mice werereceived from the Jackson Laboratory (stock number 005557).

Tumor implantation: NALM6-luc cells were grown and expanded in vitro inRPMI supplemented with 10% heat inactivated fetal bovine serum. Thecells were then transferred to a 15 ml conical tube and washed twicewith cold sterile PBS. NALM6-luc cells were then counted and resuspendedat a concentration of 10×10⁶ cells per milliliter of PBS. The cells wereplaced on ice and immediately (within one hour) implanted in the mice.NALM6-luc cells were injected intravenously via the tail vein in a 100μl volume, for a total of 1×10⁶ cells per mouse.

CAR T cell dosing: Mice were administered 5×10⁶ CAR T cells 7 days aftertumor implantation. Cells were partially thawed in a 37 degree Celsiuswater bath and then completely thawed by the addition of 1 ml of coldsterile PBS to the tube containing the cells. The thawed cells weretransferred to a 15 ml falcon tube and adjusted to a final volume of 10mls with PBS. The cells were washed twice at 1000 rpm for 10 minuteseach time and then counted on a hemocytometer. T cells were thenresuspended at a concentration of 50×10⁶ CAR T cells per ml of cold PBSand kept on ice until the mice were dosed. The mice were injectedintravenously via the tail vein with 100 μl of the CAR T cells for adose of 5×10⁶ CAR T cells per mouse. Eight mice per group were treatedeither with 100 μl of PBS alone (PBS), or humanized CD19 CAR T cells.

RAD001 dosing: A concentrated micro-emulsion of 50 mg equal to 1 mgRAD001 was formulated and then resuspended in D5W (dextrose 5% in water)at the time of dosing. Mice were orally dosed daily (via oral gavage)with 200 μl of the desired doses of RAD001.

PK analysis: Mice were dosed daily with RAD001 starting 7 days posttumor implantation. Dosing groups were as follows: 0.3 mg/kg, 1 mg/kg, 3mg/kg, and 10 mg/kg. Mice were bled on days 0 and 14 following the firstand last dose of RAD001, at the following time points for PK analysis:15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and24 hours.

Results:

The expansion and pharmacokinetics of RAD001 was tested in NSG mice withNALM6-luc tumors. Daily oral dosing of RAD001 alone did not have animpact on the growth of NALM6-luc tumors (FIG. 55). The pharmacokineticanalysis of RAD001 shows that it is fairly stable in the blood of tumorbearing mice (FIGS. 56A and 56B). Both the day 0 and day 14 PK analysesshow that the RAD001 concentrations in the blood is above 10 nm even 24hours after dosing at the lowest dose tested (0.3 mg/kg).

Based on these doses, huCAR19 CAR T cells were dosed with and withoutRAD001 to determine the proliferative ability of these cells. Thehighest dose used was 3 mg/kg based on the levels of RAD001 in the blood24 hours after dosing. As the concentration of RAD001 was above 10 nM 24hours after the final dose of RAD001, several lower doses of RAD001 wereused in the in vivo study with CAR T cells. The CAR T cells were dosedIV one day prior to the start of the daily oral RAD001 dosing. Mice weremonitored via FACS for T cell expansion.

The lowest doses of RAD001 show an enhanced proliferation of the CAR Tcells (FIG. 57). This enhanced proliferation is more evident andprolonged with the CD4⁺ CAR T cells than the CD8⁺ CAR T cells. However,with the CD8⁺ CAR T cells, enhanced proliferation can be seen at earlytime points following the CAR T cell dose.

Example 10: Anti-Tumor Activity of CD33 CAR Transduced T Cells In Vivo

HL-60 is a human acute promyelocytic leukemia cell line isolated fromthe peripheral blood of a 36 Caucasian female AML patient, and can begrown as a xenograft in immune compromised mice. This xenograft mimicsdisease in the bone marrow as seen in humans, establishing a model withwhich to test the efficacy of therapies on AMLs in the bone. These micecan be used to test the efficacy of chimeric antigen receptor (CAR) Tcells specific for cellular markers found on acute myeloid (orpromyelocytic) leukemia cells, such as CD33 (Siglec-3).

HL-60 cells were tagged with a firefly luciferase reporter gene and usedin an orthotopic model of acute myeloid leukemia (AML) inNOD.Cg-Prkdc^(scid)Il2rg^(tmIWjl)/SzJ (NSG) mice to test the efficacy ofCART cells specific for CD33.

CD33 expression was tested on HL-60 cells, and these cells were used inin vitro assays to look at the ability of CD33-specific CAR T cells torecognize and respond to the target. In vivo HL-60 cells grew whenimplanted intravenously via the tail vein, and growth was limitedprimarily to the bone marrow. One week after the tumor cells wereimplanted, the disease shifted fully to the bones and began to grow atan exponential rate. Left untreated, mice would start to displayclinical symptoms and hind limb paralysis 4-6 weeks after tumorimplantation. Several CD33 scFv clones from an in vitro screen weretested in this in vivo model.

Materials and Methods:

HL-60 cell line: The HL-60 human AML cell line was developed from theperipheral blood of a patient with acute promyelocytic leukemia. Thecells were then tagged with firefly luciferase. These suspension cellsgrew in RPMI supplemented with 10% heat inactivated fetal bovine serum.

Mice: 6 week old NSG (NOD.Cg-Prkdc^(scid)Il2rg^(tmIWjl)/SzJ) mice werereceived from the Jackson Laboratory (stock number 005557).

Tumor implantation: HL-60-luc cells were grown and expanded in vitro inRPMI supplemented with 10% heat inactivated fetal bovine serum. Thecells were then transferred to a 50 ml conical tube and washed twicewith cold sterile PBS. HL-60-luc cells were then counted and resuspendedat a concentration of 10×10⁶ cells per milliliter of PBS. The cells wereplaced on ice and immediately (within one hour) implanted in mice.HL-60-luc cells were injected intravenously via the tail vein in a 100μl volume, for a total of 1×10⁶ cells per mouse.

CAR T cell dosing: Mice were administered 5×10⁶ CAR′ T cells 14 daysafter tumor implantation. The implanted cells were growing slowly in themice; by 14 days they had started to increase the rate of tumor growth.Cells were partially thawed in a 37 degree Celsius water bath and thencompletely thawed by the addition of 1 ml of cold sterile PBS to thetube containing the cells. The thawed cells were transferred to a 15 mlfalcon tube and adjusted to a final volume of 10 mls with PBS. The cellswere washed twice at 1000 rpm for 10 minutes each time and then countedon a hemocytometer. The CAR T cells were normalized for CAR transductionso that each group has the same percentage of CAR′ T cells. The 5×10⁶CAR′ T cells were then resuspended at a concentration of 50×10⁶ CAR′ Tcells per ml of cold PBS and kept on ice until the mice were dosed. Themice were injected intravenously via the tail vein with 100 μl of theCAR T cells for a dose of 5×10⁶ CAR′ T cells per mouse. Eight mice pergroup were treated either with 100 μl of PBS alone (PBS), CD19-CARcontrol T cells (CD19), CD33-1 (clone 1) CAR T cells, CD33-2 (clone 2)CAR T cells, CD33-4 (clone 4) CAR T cells, CD33-5 (clone 5) CAR T cells,CD33-6 (clone 6) CAR T cells, CD33-7 (clone 7) CAR T cells, and CD33-9(clone 9) CAR T cells. The T cells were all prepared from the same humandonor in parallel.

Animal monitoring: The health status of the mice was monitored daily,including twice weekly body weight measurements. The percent change inbody weight was calculated as(BW_(current)−BW_(initial))/(BW_(initial))×100%. Tumor burden wasmonitored twice weekly by bioluminescent imaging. Mice wereintraperitoneally injected with D-luciferin 10 minutes prior toanesthetizing and imaging the mice with a Xenogen. Disease burden wascalculated by calculating the bioluminescence of the tumor cells(photons/second).

Results:

The anti-tumor activity of the seven CD33 CARs were evaluated anddirectly compared against CART cells directed against CD19 in the HL-60model of human acute myelogenous leukemia (AML). Following tumorimplantation on day 0, mice were randomized into treatment groups andtreated with 5×10⁶ CAR⁺ T cells intravenously on day 7. AML diseaseburden and animal health were monitored until animals achieved endpoint.The mice in the control PBS and CD19 CAR T cell groups were euthanizedon day 22 post-CAR T cell dosing (29 days post-tumor implantation) whendisease burden in the control groups was nearing maximum luminescencevia imaging. The mice in the CD33-1, CD33-2, CD33-7, and CD33-9 CAR Tcell treated groups were euthanized on day 29 post-CAR T cell dosing (36days post-tumor implantation) when disease burden in these groups hadreached the luminescence at which the control groups were euthanized.The mice in the CD33-4, CD33-5, and CD33-6 CAR T cell treated groupsshowed a late decrease in disease burden.

The bioluminescence imaging result of this study is shown in FIG. 58.Mean bioluminescence (+/−SEM) of the tumor cells showed disease burdenin the whole animal. This was shown as photons/second (p/s) of the ROI(region of interest), which was the whole mouse. The PBS treatmentgroup, which did not receive any T cells, demonstrated baseline HL-60tumor growth kinetics in intravenously implanted NSG mice. The CD19treatment group received control CAR T cells, not specific for HL-60cells, which underwent the same in vitro expansion process as the CD33CAR T cells. These cells served as a T cell control to show thenon-specific response of the T cells in this tumor model. Both the PBSand the CD19 CAR T cell treatment groups demonstrated continuous tumorprogression throughout the experiment. All of the CD33 CART cellsdelayed the progression of disease after the 5×10⁶ CAR T cellinjections, though there appeared to separation of the clones into twogroups with a differential response.

The anti-tumor activity of CD33 CAR transduced T cells was assessed inthis efficacy study in NSG mice bearing a xenograft model of human AML.These studies show that the HL-60-luc model recapitulated human AML, inthe NSG mouse and was capable of being targeted by CD33 CAR T cells(FIG. 58). The growth of the HL-60-luc human AML xenograft in NSG miceafter treatment with CAR T cells specific for CD33 demonstrates a delayin disease progression (FIG. 58). This study demonstrated that sevenCD33 CARs were capable of mounting an anti-tumor response in a xenograftmodel of AML (FIG. 58).

EQUIVALENTS

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific aspects, it is apparent that other aspects and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such aspects andequivalent variations.

1-64. (canceled)
 65. A method of providing an anti-tumor immunity in amammal, comprising administering to the mammal an effective amount of acell comprising a chimeric antigen receptor (CAR) polypeptide, or anucleic acid encoding the CAR polypeptide, wherein the CAR polypeptidecomprises a CD33 binding domain, a transmembrane domain, and anintracellular signaling domain comprising a costimulatory domain, aprimary signaling domain, or both a costimulatory domain and a primarysignaling domain, and wherein said CD33 binding domain comprises a heavychain variable region and a light chain variable region selected fromthe group consisting of: (i) a heavy chain variable region comprising: aheavy chain complementary determining region 1 (HC CDR1) comprising thesequence of SEQ ID NO: 274, a heavy chain complementary determiningregion 2 (HC CDR2) comprising the sequence of SEQ ID NO: 283, and aheavy chain complementary determining region 3 (HC CDR3) comprising thesequence of SEQ ID NO:292 present in order of HC CDR1, HC CDR2, and HCCDR3; and a light chain variable region comprising: a light chaincomplementarity determining region 1 (LC CDR1) comprising the sequenceof SEQ ID NO: 301, a light chain complementarity determining region 2(LC CDR2) comprising the sequence of SEQ ID NO: 310, and a light chaincomplementarity determining region 3 (LC CDR3) comprising the sequenceof SEQ ID NO:319 present in order of LC CDR1, LC CDR2, and LC CDR3; (ii)a heavy chain variable region comprising: a heavy chain complementaritydetermining region 1 (HC CDR1) comprising the sequence of SEQ ID NO:328, a heavy chain complementarity determining region 2 (HC CDR2)comprising the sequence of SEQ ID NO: 337, and a heavy chaincomplementarity determining region 3 (HC CDR3) comprising the sequenceof SEQ ID NO:346 present in order of HC CDR1, HC CDR2, and HC CDR3; anda light chain variable region comprising: a light chain complementaritydetermining region 1 (LC CDR1) comprising the sequence of SEQ ID NO:355, a light chain complementarity determining region 2 (LC CDR2)comprising the sequence of SEQ ID NO: 364, and a light chaincomplementarity determining region 3 (LC CDR3) comprising the sequenceof SEQ ID NO:373 present in order of LC CDR1, LC CDR2, and LC CDR3; and(iii) a heavy chain variable region comprising: a heavy chaincomplementarity determining region 1 (HC CDR1) comprising the sequenceof SEQ ID NO: 89, a heavy chain complementarity determining region 2 (HCCDR2) comprising the sequence of SEQ ID NO: 98, and a heavy chaincomplementarity determining region 3 (HC CDR3) comprising the sequenceof SEQ ID NO:107, present in order of HC CDR1, HC CDR2, and HC CDR3; anda light chain variable region comprising: a light chain complementaritydetermining region 1 (LC CDR1) comprising the sequence of SEQ ID NO:116, a light chain complementarity determining region 2 (LC CDR2)comprising the sequence of SEQ ID NO: 125, and a light chaincomplementarity determining region 3 (LC CDR3) comprising the sequenceof SEQ ID NO:134 present in order of LC CDR1, LC CDR2, and LC CDR3, 66.A method of treating a mammal having a disease associated withexpression of CD33, comprising administering to the mammal an effectiveamount of a cell, comprising a chimeric antigen receptor (CAR)polypeptide, or a nucleic acid encoding the CAR polypeptide, wherein theCAR comprises a CD33 binding domain, a transmembrane domain, and anintracellular signaling domain comprising a costimulatory domain, aprimary signaling domain, or both a costimulatory domain and a primarysignaling domain, and wherein said CD33 binding domain comprises a heavychain variable region and a light chain variable region selected fromthe group consisting of: (i) a heavy chain variable region comprising: aheavy chain complementary determining region 1 (HC CDR1) comprising thesequence of SEQ ID NO: 274, a heavy chain complementary determiningregion 2 (HC CDR2) comprising the sequence of SEQ ID NO: 283, and aheavy chain complementary determining region 3 (HC CDR3) comprising thesequence of SEQ ID NO:292 present in order of HC CDR1, HC CDR2, and HCCDR3; and a light chain variable region comprising: a light chaincomplementarity determining region 1 (LC CDR1) comprising the sequenceof SEQ ID NO: 301, a light chain complementarity determining region 2(LC CDR2) comprising the sequence of SEQ ID NO: 310, and a light chaincomplementarity determining region 3 (LC CDR3) comprising the sequenceof SEQ ID NO:319 present in order of LC CDR1, LC CDR2, and LC CDR3; (ii)a heavy chain variable region comprising: a heavy chain complementaritydetermining region 1 (HC CDR1) comprising the sequence of SEQ ID NO:328, a heavy chain complementarity determining region 2 (HC CDR2)comprising the sequence of SEQ ID NO: 337, and a heavy chaincomplementarity determining region 3 (HC CDR3) comprising the sequenceof SEQ ID NO:346 present in order of HC CDR1, HC CDR2, and HC CDR3; anda light chain variable region comprising: a light chain complementaritydetermining region 1 (LC CDR1) comprising the sequence of SEQ ID NO:355, a light chain complementarity determining region 2 (LC CDR2)comprising the sequence of SEQ ID NO: 364, and a light chaincomplementarity determining region 3 (LC CDR3) comprising the sequenceof SEQ ID NO:373 present in order of LC CDR1, LC CDR2, and LC CDR3; and(iii) a heavy chain variable region comprising: a heavy chaincomplementarity determining region 1 (HC CDR1) comprising the sequenceof SEQ ID NO: 89, a heavy chain complementarity determining region 2 (HCCDR2) comprising the sequence of SEQ ID NO: 98, and a heavy chaincomplementarity determining region 3 (HC CDR3) comprising the sequenceof SEQ ID NO:107, present in order of HC CDR1, HC CDR2, and HC CDR3; anda light chain variable region comprising: a light chain complementaritydetermining region 1 (LC CDR1) comprising the sequence of SEQ ID NO:116, a light chain complementarity determining region 2 (LC CDR2)comprising the sequence of SEQ ID NO: 125, and a light chaincomplementarity determining region 3 (LC CDR3) comprising the sequenceof SEQ ID NO:134 present in order of LC CDR1, LC CDR2, and LC CDR3 67.The method of claim 66, wherein the CAR polypeptide is encoded by anucleic acid comprising the sequence of SEQ ID NO:
 80. 68. The method ofclaim 65, wherein the CAR polypeptide comprises a light chain variableregion selected from the group consisting of: (i) the amino acidsequence of SEQ ID NO: 71; and (ii) an amino acid sequence with 95-99%identity to the amino acid sequence of SEQ ID NO:
 71. 69. The method ofclaim 65, wherein the CAR polypeptide comprises a heavy chain variableregion selected from the group consisting of: (i) the amino acidsequence of SEQ ID NO: 62 (ii) an amino acid sequence with 95-99%identity to the amino acid sequence of SEQ ID NO:
 62. 70. The method ofclaim 65, wherein the CAR polypeptide comprises a single chain variablefragment (scFv) selected from the group consisting of: (i) the aminoacid sequence of SEQ ID NO: 44 or 266; (ii) an amino acid sequence with95-99% identity to SEQ ID NO: 44 or
 266. 71. The method of claim 65,wherein the transmembrane domain of the CAR polypeptide comprises anamino acid sequence selected from the group consisting of: (i) the aminoacid sequence of SEQ ID NO: 6; and (ii) an amino acid sequence with95-99% identity to the amino acid sequence of SEQ ID NO:6.
 72. Themethod of claim 65, wherein the costimulatory domain of the CARpolypeptide comprises the amino acid sequence of SEQ ID NO:7, or asequence with 95-99% identity to the amino acid sequence of SEQ ID NO:7.73. The method of claim 65, wherein, wherein the intracellular signalingdomain of the CAR polypeptide comprises a functional signaling domain of4-1BB and a functional signaling domain of CD3 zeta.
 74. The method ofclaim 65, wherein the intracellular signaling domain of the CARpolypeptide comprises an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NO: 7 and the sequenceof SEQ ID NO:9 or SEQ ID NO:10; and a sequence with 95-99% identity tothe amino acid sequence of SEQ ID NO:7 and a sequence with 95-99%identity to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. 75.The method of claim 65, wherein the CAR polypeptide comprises an aminoacid sequence selected from the group consisting of: (i) the amino acidsequence of SEQ ID NO: 53; and (ii) an amino acid sequence with 95-99%identity to SEQ ID NO:
 53. 76. The method of claim 65, wherein theprimary signaling domain of the CAR polypeptide comprises a functionalsignaling domain of CD3 zeta, wherein the CD3 zeta comprises the aminoacid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or a sequence with95-99% identity to the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO:10.
 77. The method of claim 65, wherein the intracellular signalingdomain of the CAR polypeptide comprises a costimulatory domain.
 78. Themethod of claim 65, wherein the intracellular signaling domain of theCAR polypeptide comprises a primary signaling domain.
 79. The method ofclaim 65, wherein the intracellular signaling domain of the CARpolypeptide comprises a costimulatory domain and a primary signalingdomain.
 80. The method of claim 66, wherein the disease associated withCD33 expression is: (i) a cancer or malignancy, or a precancerouscondition chosen from one or more of a myelodysplasia, a myelodysplasticsyndrome or a preleukemia, or (ii) a non-cancer related indicationassociated with expression of CD33.
 81. The method of claim 66, whereinthe disease is a hematologic cancer.
 82. The method of claim 66, whereinthe disease is an acute leukemia chosen from one or more of acutemyeloid leukemia (AML); myelodysplastic syndrome (MDS);myeloproliferative neoplasm; chronic myeloid leukemia (CML); or Blasticplasmacytoid dendritic cell neoplasm, or a combination thereof.
 83. Themethod of claim 82, wherein the MDS comprises one or more of: (a)refractory anemia; (b) refractory anemia with ring sideroblasts; (c)refractory anemia with excess blasts; (d) refractory anemia with excessblasts in transformation; or (e) chronic myelomonocytic leukemia (CML).84. The method of claim 65, wherein the effective amount of the cell isadministered in combination with one or more of: (a) a chemotherapeuticagent; (b) a bone marrow transplantation; (c) a lymphodepleting therapy;(d) an agent which inhibits PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA,TIGIT, LAIR1, CD160 or 2B4; (e) a therapy for CRS (cytokine releasesyndrome); or (f) an mTOR inhibitor.
 85. The method of claim 84, whereinthe agent comprises: (a) 5-azacytidine; (b) decitabine; (c)lenalidomide; or (d) an iron chelator wherein the iron chelator isdeferoxamine or deferasirox.
 86. The method of claim 65, wherein thecells expressing a CAR molecule are administered at a dose of 10⁴ to 10⁹cells/kg body weight, 10⁵ to 10⁶ cells/kg body weight per dose.
 87. Themethod of claim 65, wherein the mammal receives one or more subsequentadministrations the population of immune effector cells.
 88. The methodof claim 65, wherein the mammal is a human.